System and method for isolating and analyzing cells

ABSTRACT

A system and method for isolating and analyzing single cells, wherein the system includes: an array of wells defined at a substrate, each well including an open surface and a well cavity configured to capture cells in one of a single-cell format and single-cluster format, and a fluid delivery module including a fluid reservoir superior to the array of wells through which fluid flow is controlled along a fluid path in a direction parallel to the broad face of the substrate; and wherein the method includes: capturing a population of non-cell particles into the array of wells in single-particle format; releasing, from the non-cell particles, a set of probes into the array of wells; capturing a population of cells into the array of wells in single-cell format; releasing biomolecules from each captured cell into the array of wells; and generating a set of genetic complexes comprising the biomolecules associated with a single captured cell and a subset of probes within individual wells of the array of wells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/115,370, filed 28 Aug. 2018, which claims the benefit of U.S.Provisional Application No. 62/551,575 filed on 29 Aug. 2017 and U.S.Provisional Application No. 62/671,750 filed on 15 May 2018, which areboth incorporated in their entirety herein by this reference.

TECHNICAL FIELD

This invention relates generally to the cell sorting and analysis field,and more specifically to a new and useful system and method forcapturing and analyzing cells within the cell sorting field.

BACKGROUND

With an increased interest in cell-specific drug testing, diagnosis, andother assays, systems that allow for individual cell isolation,identification, and retrieval are becoming more desirable within thefield of cellular analysis. Furthermore, with the onset of personalizedmedicine, low-cost, high fidelity cellular sorting and geneticsequencing systems are becoming highly desirable. However, conventionaltechnologies for cell capture systems posses various shortcomings thatprevent widespread adoption for cell-specific testing. For example, flowcytometry requires that the cell be simultaneously identified andsorted, and limits cell observation to the point at which the cell issorted. Flow cytometry fails to allow for multiple analyses of the samecell within a single flow cytometry workflow, and does not permitarbitrary cell subpopulation sorting. In other examples, conventionalmicrofluidic devices rely on cell-specific antibodies for cellselection, wherein the antibodies that are bound to the microfluidicdevice substrate selectively bind to cells expressing the desiredantigen. Conventional microfluidic devices can also fail to allow forsubsequent cell removal without cell damage, and only capture the cellsexpressing the specific antigen; non-expressing cells, which could alsobe desired, are not captured by these systems. Such loss of cellviability can preclude live-cell assays from being performed on sortedor isolated cells. Cellular filters can separate sample components basedon size without significant cell damage, but suffer from clogging and donot allow for specific cell identification, isolation of individualcells, and retrieval of identified individual cells. Other technologiesin this field are further limited in their ability to allow multiplexassays to be performed on individual cells, while minimizing samplepreparation steps and overly expensive instrumentation.

In the field of single cell analysis, the isolation, identification andgenetic analysis of rare cells, such as cancer stem cells, currentlysuffer limitations in accuracy, speed, and throughput. Furthermore, manysystems do not maintain the viability and/or quality of living cells orbiological materials extracted from cells, as typical methods foridentification of cells during the isolation process necessitatesfixation, staining, or an additional biochemical process at highertemperatures, which may damage the cell and/or its genetic material, inaddition to slowing processing speed. Thus, there is a need in the cellsorting field to create new and useful systems and methods for isolatingand analyzing cells, which are able to maximize viability of cells andtheir intracellular components, including biomolecules such as messengerRNA, for downstream analysis. Furthermore, cell isolation workflows thatfurther include molecular indexing of biomolecules and processing ofgenetic transcripts can provide several benefits for improvingthroughput and accuracy for applications in cellular analysis, includingmassively parallel RNA sequencing for full-length mRNA, whole genomesand/or single-cell exomes. To date, there are no systems and/or methodsthat facilitate single cell isolation and DNA/RNA sequencing libraryconstruction on a single, unified device. The system and methoddescribed herein address these limitations by integrating functions suchas single-cell capture, biomolecule labeling, fluid delivery, andtemperature modulation, in order to enable more advanced biochemicalprocesses to be performed on individual cells within the same array ofwells used to capture the cells (e.g., reverse transcription, polymerasechain reaction, single cell genome (DNA/RNA) sequencing), thereby vastlyimproving capture efficiency for desired cells and increasing speed andanalytical capabilities for single-cell experimental workflows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of an embodiment of a portion of asystem for isolating and analyzing cells;

FIGS. 2A-2C depict variations of a portion of a system for isolating andanalyzing cells;

FIGS. 3A-3B depict variations of a portion of a system for isolating andanalyzing cells;

FIG. 4A depicts a variation of a portion of a system for isolating andanalyzing cells;

FIGS. 4B-4E depict schematic representations of example configurationsof a portion of a system for isolating and analyzing cells;

FIGS. 5A-5C depict variations of a portion of a system for isolating andanalyzing cells;

FIGS. 6A-6B depict variations of a portion of a system for isolating andanalyzing cells;

FIG. 7 depicts an example of a variation of a portion of a system forisolating and analyzing cells;

FIGS. 8A-8B depict a variation of a portion a system for isolating andanalyzing cells;

FIGS. 9A-9B depict schematic representations of example configurationsof a portion of a system for isolating and analyzing cells;

FIG. 10 depicts a schematic representation of a variation of a portionof a system for isolating and analyzing cells;

FIGS. 11A-11B depict a schematic representation of a variation of aportion of a system for isolating and analyzing cells;

FIGS. 12A-12B depict a variation of a portion of a system for isolatingand analyzing cells;

FIG. 13A depicts an variation of a portion of a system for isolating andanalyzing cells;

FIG. 13B depicts a variation of a portion of a system for isolating andanalyzing cells;

FIG. 14 depicts a variation of a portion of a system for isolating andanalyzing cells;

FIG. 15A-15B depict cross-sectional views of a variation of a portion ofa portion of a system for isolating and analyzing cells;

FIG. 16 depicts a variation of a system for isolating and analyzingcells;

FIG. 17 depicts a variation of a portion of a system for isolating andanalyzing cells;

FIG. 18 depicts a flow chart for an embodiment of a method for isolatingand analyzing cells;

FIG. 19 depicts a schematic representation of a variation of an portionof a method for isolating and analyzing cells;

FIG. 20 depicts a schematic representation of a variation of a portionof an embodiment of a method for isolating and analyzing cells;

FIG. 21 depicts a schematic representation of a variation of a portionof an embodiment of a method for isolating and analyzing cells;

FIG. 22 depicts a schematic representation of a variation of a portionof an embodiment of a method for isolating and analyzing cells;

FIG. 23 depicts a schematic representation of a variation of a portionof an embodiment of a method for isolating and analyzing cells;

FIG. 24 depicts a flow chart for an embodiment of a method for isolatingand analyzing cells;

FIG. 25 depicts a schematic representation of a variation of a portionof an embodiment of a method for isolating and analyzing cells;

FIG. 26 depicts a schematic representation of a variation of a portionof an embodiment of a method for isolating and analyzing cells;

FIG. 27 depicts an example of an embodiment of a portion of a system anda method for isolating and analyzing cells;

FIG. 28A-28C depicts an example of an embodiment of a portion of asystem for isolating and analyzing cells;

FIG. 29 depicts an example of a variation of a portion of a system forisolating and analyzing cells;

FIG. 30A-30B depict an example of a variation of a portion of a systemfor isolating and analyzing cells;

FIG. 31A-31C depict an example of a variation of a portion of a systemfor isolating and analyzing cells;

FIG. 32A-32C depicts an example of a variation of a portion of a systemfor isolating and analyzing cells;

FIG. 33 depicts an example of a variation of a portion of a system forisolating and analyzing cells;

FIG. 34 depicts a schematic of three variations of a portion of anembodiment of a method for isolating and analyzing cells;

FIG. 35 depicts an embodiment of a system for isolating and analyzingcells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the inventionis not intended to limit the invention to these preferred embodiments,but rather to enable any person skilled in the art to make and use thisinvention.

1. System

As shown in FIG. 1, a system 100 for isolating and analyzing a set ofcells comprises: a substrate 110 having a broad surface; an array ofwells 120 defined at a first side 112 (e.g., upper broad surface) of thesubstrate, each well 128 in the array of wells 120 including an opensurface 122 defined at the first side 112, a base surface 124 definedwithin the substrate proximal a second side 114 (e.g., lower broadsurface) directly opposing the first side 112, and a set of walls 126extending between the base surface 124 and the open surface 122 to forma well cavity 128 of the well 128. The array of wells 120 can bearranged in an active region 116 of the substrate, or in any othersuitable arrangement. To facilitate sample or fluid 40 delivery to thearray of wells 120, the system 100 can further include a fluid deliverymodule 140 configured to couple to the substrate 110 and transfer asample containing the set of cells and/or another fluid 40 to the arrayof wells 120. Additionally or alternatively, the system 100 can includea flow control subsystem (e.g., pressure pump system) 180 configured tocontrol fluid 40 (e.g., biological sample, process reagent, solutioncontaining non-cell particles) flow (e.g., direction, velocity, volumeof the fluid) through the system 100, as well as any other suitable flowthrough the system requiring control and/or actuation. Additionally oralternatively, the system 100 can include a thermal control module 190for controlling the temperature of portions of the system 100.Additionally or alternatively, the system 100 can include an imagingsubsystem 194 configured to perform optical imaging, illumination, orirradiation of the contents of the array of wells 120, and to enableidentification, localization, and quantification of cells retained bywells of the array of wells 120. Additionally or alternatively, thesystem 100 can include an extraction module that can extract one ormore: target cells, particles, cell-particle pairs, genetic complexes,and/or genetic products from the array of wells. However, variations ofthe system 100 can include any other suitable component in any suitableconfiguration and/or combination, as described in U.S. application Ser.No. 15/333,420 entitled “Cell Capture System and Method of Use” andfiled 25 Oct. 2016, U.S. application Ser. No. 14/208,298 entitled“System for capturing and analyzing cells” and filed 13 Mar. 2014, andU.S. application Ser. No. 14/289,155 entitled “System and Method forIsolating and Analyzing Cells” and filed 28 May 2014, which are eachincorporated in their entirety by this reference.

The system 100 functions to isolate, capture, retain, and analyze cellsof a cell population 20, in at least one of single-cell format andsingle-cluster format, at known, addressable locations, and further tofacilitate performance of multiple single-cell assays that can beperformed on individual target cells (e.g., rare cells in a biologicalsample) or clusters of cells (e.g., doublets, triplets). In preferredembodiments, the system 100 functions to facilitate the preparation ofgenetic libraries (e.g., cDNA generated from captured mRNA from lysedtarget cells, amplified cDNA, amplified DNA) of captured single cellsfor sequencing proximately following, and within the same device, ascell capture. Once cells are captured in defined locations determined bysingle cell capture wells, the fluid delivery module of the system 100can be used to provide and deliver reagents simultaneously,sequentially, and/or in repetition to enable a variety of cellular,sub-cellular or molecular reactions to be performed in each of thesingle cells/cell clusters. Additionally or alternatively, the system100 can function to capture and process non-cell particles (e.g.,nucleic acid material, other biological material, other non-biologicalmaterial, particles containing molecular probes or reagents, etc.), aswell as combinations of single cells and single non-cell particles(e.g., a cell-particle pair). Furthermore, the system 100 can enablecontrolled and rapid thermal modulation of the array of wells andadditionally or alternatively of the fluid 40 delivered to the array ofwells (e.g., heating and cooling cycles from 95° C. to 5° C.), tomaintain cell or biological material viability, increase efficiency ofbiological assays, and perform a variety of biochemical processes withinthe set of wells. The system 100 can also allow optical interrogationand detection of events on each of the captured cells at a singlecell/single cluster level. The system 100 can additionally oralternatively enable selective release and/or selective removal of oneor more of the captured cells or non-cell particles for furtherprocessing and analysis. In some embodiments, the system 100 can conferthe benefits of real-time cell tracking, viable cell retrieval,biochemical processes (e.g., cell lysis, cell fixation, polymerase chainreaction, reverse transcription, etc.) and selective downstreammolecular analysis (e.g., electrophoresis, sequencing, fluorescenceimaging), either in the same microfluidic chip or off-chip. In someembodiments, the system 100 can be used to capture circulating tumorcells (CTCs) and subpopulations of CTCs, such as circulating stem cells(CSCs), but can additionally or alternatively be used to capture anyother suitable cell (e.g., erythrocytes, monocytes, macrophages,osteoclasts, dendritic cells, microglial cells, T-cells, B-cells,megakaryocytes, germ cells, nurse cells, neural cells, stem cells, etc.)or biological material of possible interest. The system 100 ispreferably defined on a substrate 110, more preferably a microfluidicchip, but can alternatively be located on or defined by any suitablesubstrate.

In specific examples, the system 100 can be used with method(s) operablefor single cell polymerase chain reaction (PCR), wherein such systemscan facilitate high efficiency capture of cells (e.g., 100s, of cells,1000s of cells, 10,000s of cells, 100,000s of cells, 1,000,000 of cells,etc.) in single cell format (or single cluster format) within wells, aswell as on-chip reagent delivery to the wells, incubation, andthermocycling in order to provide a cell capture-to-PCR workflow. Inmore detail, microfluidic and other portions of the system can beoperable to perform assays (e.g., assays associated with ARV7 mRNA)using PCR with sample (e.g., prostate clinical samples) with single cellor single cell cluster resolution. In specific examples, the system 100can accommodate sample volumes as low as 10 μl to as high as on theorder of up to 1 mL within a fluid reservoir 160 associated with thearray of wells 120, wherein the sample can contain a range of between500 to 100,000 target cells, thereby providing the ability to processlarger sample volumes containing a large number of cells of interest.

The system 100 preferably achieves individual cell capture and retentionfrom a biological sample including a cell population 20 without antibodycoated wells, and preferably maintains the viability of the cellsthroughout isolation, capture, retention, and/or removal. Individualcell capture is preferably achieved by flowing or dispensing a samplecontaining a group of single cells within a fluid layer over the arrayof wells 120 in a direction parallel (e.g., substantially parallel,within 0.1 degrees of parallel, within 1 degree of parallel, within 45degrees of parallel, completely parallel, etc.) to the broad surface ofthe substrate, and capturing the cells once they have descended throughthe fluid layer towards the array of wells 120 under the influence ofgravity. Alternatively, individual cell capture can be achieved bydelivering a sample containing a group of single cells into a fluidlayer provided by a fluid reservoir 160, over the array of wells 120 ina direction perpendicular to the broad surface of the substrate, andcapturing the cells once they have descended through the fluid layertowards the array of wells 120 under the influence of gravity. However,in some variations, individual cell capture can additionally oralternatively be achieved by any suitable mechanism for promoting singlecell transfer into a well of the set of wells. Furthermore, the system100 is preferably configured to prevent undesired fluid currents thatcan lift cells from the substrate or move cells/cell clusters from wellcavities 128 at which the cells are captured and fully retained within.However, in some variations, the system 100 can be configured tofacilitate moving of cells/cell clusters in any suitable manner. Theflow path of a fluid (e.g., biological sample, process reagent 40)through the system 100 is preferably multi-directional and uniform, suchthat each cell/cell cluster in the system 100 experiences consistentconditions (e.g., gradient length scales along the flow path of flowproperties such as pressure, density, temperature, solution composition,and other suitable properties are large relative to the length scales ofthe system); however, the flow path can alternatively be unidirectional,bi-directional, or have any other suitable characteristic(s). Invariations of a specific example, as shown in FIG. 2A-2C, the flow path141 of a fluid through the system includes a set of fluid pathways 146(e.g., of a manifold coupled to the array of wells) of equal length(e.g., substantially equal length, equal length to withinmanufacturability tolerances, etc.) that are configured such that areagent supplied at a manifold inlet 440 to the set of fluid pathways146 arrives at each array of inlets 444 (e.g., a single well, along aregion of a first edge of the reservoir, along region of a first edge ofthe active region of the substrate, etc.) at substantially the same timepoint (e.g., at the same time, within 1 second, within 1 minute, etc.),and passing across the active region 116 of the substrate (e.g.,containing the array of wells 120) through an array of outlets 445 to amanifold outlet 442. Cell transport, isolation, sorting and viabilitymaintenance can additionally be accomplished by controlling the sampleflow rate through the system (e.g., by adjusting the flow rate so that acharacteristic length scale of the flow is of a similar order as acharacteristic length scale of a well, by dithering the flow ratebetween high and low flow conditions, etc.), or through any othersuitable means. However, the flow characteristics of a fluid throughsystem 100 may be otherwise configured.

In operation, the system 100 preferably receives a biological sampleincluding the cell population 20 and facilitates distribution of thebiological sample uniformly across the array of wells 120 (e.g., usinguniform cross flow, smearing, a cytospin procedure, pipetting aliquotsof the sample at different regions of the array etc.). However, thesystem 100 can additionally or alternatively facilitate distribution ofthe fluid 40 (e.g., biological sample, process reagent, non-cellparticles) across the set of wells using positive pressure (e.g.,positive pressure at an inlet to the array) and/or negative pressure(e.g., negative pressure at an outlet of the array) applied by the flowcontrol subsystem 180. Additionally or alternatively, actuation pressurethat facilitates sample distribution can be cycled in a pulse-widthmodulation fashion or sinusoidal fashion to provide net actuationpressure, either net positive at the inlet or net negative at theoutlet. As such, desired cells having a defining characteristic (e.g.,size-based characteristic, density-based characteristic, adhesion-basedcharacteristic, etc.) can be trapped within a well 128 as the biologicalsample flows across the array of wells 120. For example, in thevariation of the system 100 configured to capture CTCs, the wells arepreferably configured based upon defining morpohological features of CTCcells, in order to facilitate capture and retention of CTCs in singlecell or single cluster format. However, the system 100 can additionallyor alternatively be configured to retain and facilitate processing orany other suitable particle of interest in any other suitable format.Actuation pressure is preferably provided by the flow control subsystem180 (e.g., a manually-operated pipette, automated fluid-handling robot,vacuum pressure system, electromechanical micropump, etc.) in fluidcommunication with the system 100, but can alternatively or additionallybe provided by any suitable mechanism.

In a preferred embodiment of the system 100, shown in FIG. 35, up to twoindividual arrays of wells can be processed in parallel (synchronously,asynchronously). However, the components of the system can be configuredwith any numerosity to accomadate any suitable number of arrays.

1.1 System—Substrate

As shown in FIG. 3, the substrate 110 functions to provide a medium atwhich the array of wells 120 (set of microwells, microwells, wells) canbe defined. In variations, the substrate 110 can have a first side(e.g., upper broad surface) 112, and a second side (e.g., lower broadsurface) directly opposing the first side. The upper broad surface 112of the substrate 110 is preferably a planar surface, such thatmicrofluidic elements (e.g., inlet, outlet, inlet manifold, outletmanifold, fluid channels, etc.) of the system 100 are defined at leastpartially at a planar surface. Alternatively, the upper broad surface112 of the substrate 110 can be a non-planar surface, such thatmicrofluidic elements of the system 100 are defined at least partiallyat a non-planar surface. In variations, the non-planar surface can be aconcave surface, a convex surface, or a surface having concave, planar,and/or convex surfaces. Such variations can facilitate various methodsof depositing and distributing a sample at the array of wells 120. Inany variations of the substrate 110 including a non-planar upper broadsurface 112, the non-planar portion(s) are preferably shallow (e.g.,having a small depth relative to a width of the broad surface) or short(e.g., having a small height relative to a width of the broad surface);however, the non-planar portion(s) can additionally or alternativelyinclude portions that are deep (e.g., having a large depth relative to awidth of the broad surface) or tall (e.g., having a large heightrelative to a width of the broad surface). However, the surface canalternatively have any other suitable axis or type of symmetry, or canbe asymmetrical. In a preferred application, the first side of thesubstrate can define an alignment axis for a surface plane 118, whereinthe surface plane 118 is parallel and coaxial to the first side of thesubstrate, and aligned between the first side of the substrate, and thebase of a fluid reservoir through which fluids, such as a sample orprocess reagent, may flow to access the array of wells at the substrate.

The substrate 110 composition can provide desired characteristicsrelating to any one or more of: mechanical characteristics (e.g.,substrate mechanical properties as a mechanical stimulus), opticalproperties (e.g., transparency), electrical properties (e.g.,conductivity), thermal properties (e.g., conductivity, specific heat,etc.), physical characteristics (e.g., wettability, porosity, etc.), andany other suitable characteristic. The substrate 110 is preferablycomposed of a rigid material with high transparency (e.g., a transparentmaterial, a translucent material), in order to facilitate imaging of thesubstrate 110 to analyze captured single cells/cell clusters. The hightransparency material is preferably optically transparent, but canadditionally or alternatively be transparent and/or translucent to otherportions of the electromagnetic spectrum (e.g., microwaves, nearinfra-red, ultraviolet, etc.) In a few such variations, the substrate110 can be composed of any one or more of: glass, ceramic, asilicone-based material (e.g., polydimethylsiloxane (PDMS)), a polymer(e.g., agarose, polyacrylamide, polystyrene, polycarbonate, poly-methylmethacrylate (PMMA), polyethylene glycol, etc.), paper, a porousmaterial, and any other suitable material, including composites thereof,with high transparency. Alternatively, the substrate 110 can be composedof any other suitable material having any other suitable opticalproperties. Additionally or alternatively, the substrate can be composedof any one or more of: a ceramic material, a semi-conducting material, apolymer, and any other suitable material.

The substrate 110 can be processed using any one or more of: etchingmethods, molding methods, printing methods (e.g., 3D printingprocesses), machining methods, and any other suitable manufacturingprocesses suited to a brittle, elastic, or ductile substrate material.Furthermore, features defined at the upper broad surface 112, includingthe array of wells, can be produced by any one or more of: molding, bypolishing, by spinning a material in a flow phase followed by settingthe material, by machining, by printing (e.g., 3D printing), by etching,and by any other suitable process. In a specific example, the array ofwells 120 is defined within a silicon mold using a three maskphotolithographic process and deep reactive ion etching (DRIE) processto etch microfluidic elements into the silicon mold. In the specificexample, the etched elements of the silicon mold are then transferredpolymethylmethacrylate (PMMA) sheets as a substrate 110 using a hotembossing process. The substrate 110 in the specific example hasdimensions of 3 inches by 1 inch, in order to substantially matchdimensions of a glass microscope slide. In variations of the specificexample, and/or for other variations of the array of wells 120, hotembossing of cyclic olefin polymer (COP) can be substituted for PMMA toform the microfluidic structures of the array of wells 120. However, thesubstrate 110 can alternatively be any other suitable substrate 120processed in any other suitable manner.

Preferably, the substrate includes features that permit interaction(e.g., reversible or non-reversible attachment, coupling) to othersubcomponents of system 100. In one variation, the substrate 110 can becoupled to components the fluid delivery module 140, wherein thesubstrate includes an inlet 142 and an outlet 144 to transmit fluid 40into and out of the active region 116 of the substrate. In an example ofthis variation, the set of inlet channels of the inlet manifold and theset of outlet channels of the outlet manifold 164 can be embeddeddirectly within the substrate between the upper and lower broadsurfaces, but can additionally or alternatively be fabricated into atleast a portion of the upper broad surface of the substrate. In anotherexample, as shown in FIGS. 28A-28C, FIG. 29, and FIGS. 30A and 30B, thesubstrate 110 can be aligned to a first plate 150 containing a recess152 superior to the active region of the substrate, wherein, uponattachment of the first plate 150 to the substrate, a region of thefirst plate 150 can be aligned above the array of wells to cooperativelydefine a fluid reservoir 160 to transfer fluid across the array of wellsduring operation of system 100. The fluid reservoir 160 can be sealed bya reservoir lid 164 that can be reversibly attached to the first plate150, such that the combined assembly provides a fluid pathway 162 fordelivery of reagents, air, oil or other materials to flow parallel tothe surface of the array of wells. The resealable reservoir lid 164 caninclude a set of grooves 165 at the base of the reservoir lid, at theregion of the reservoir lid that is inserted into the fluid reservoir,for ease of displacement of immiscible liquid (water replacing oil, oilreplacing water) and, additionally or alternatively, displacement ofdifferent fluidic phases (water replacing air or air displacing water)along the fluid pathway 162. The grooves 165 of the reservoir lid 164can posses characteristic dimensions on the order of: 25 microns, 50microns, 100 microns, 150 microns, 200 microns, 250 microns, 300microns, 350 microns, 400 microns, 500 microns and/or 1 millimeter. Thenumber of grooves and dimensions of grooves 165 can be adjusted to allowfor specific dead-volume of liquid to be provided in the systems, suchas approximately: 10 microliters, 25 microliters, 50 microliters, 75microliters, 100 microliters, 150 microliters, and/or 200 microliters.The material of the lid may be optically transparent to allow imaging ofthe cells or beads captured in the microwell arrays and/orUV-irradiation for photocleaving of specific biomolecules from surfacesof the microwells, particles, and/or genetic complexes, as described inSection 2. The reservoir lid 164 also accommodates elastomeric surfaces(FIG. 33) to allow for proper seal with the fluidic manifold. Theresealable lid may be opened or closed at the beginning of the fluidicoperation, in the middle of the process or at the end of the process asneeded to deliver cells to the array of wells, deliver particles to thearray of wells, deliver reagents to the array of wells, and/or removespecific cells, particles, and/or fluids from the array of wells. Thereservoir lid 164 is designed for easy attachment or removal, eithermanually or in an automated fashion, however can be configured foroperation in any other suitable manner to provide a complete fluidicsystem.

In a second variation, the substrate 110 can be attached to a substrateplatform 105 that functions to reversibly attach and align the substrateto a platform, heating element (e.g., thermal control module 194),and/or stage upon which assays are performed, wherein the stage can beused to physically adjust the position of the substrate within thesystem 100 to improve access of the array of wells to other elements ofthe system, such as the imaging subsystem 194, thermal control module190, and/or the extraction module. Preferably, the substrate platform105 can be configured to accommodate, secure, and manipulate substrateswith various array configurations (e.g., arrays with 50,000 wells,arrays with 1M wells, etc.) with high precision, and can include anoptional substrate attachment mechanism 110. As shown in examplesdepicted in FIGS. 30A-30B, FIGS. 31A-31C, FIGS. 32A-32C, and FIG. 33,the substrate platform can include a platform lid 115 that canaccommodate and support the reservoir lid 164, wherein the platform lid115 functions to reversibly secure and seal the reservoir lid 164 intothe fluid reservoir 160 formed by the first plate 150 attached to thesubstrate. In variations, the platform lid 115 can include anelastomeric gasket or sealing element and a detent plunger that appliespressure in a range between 1-4 pounds, in order to hermetically sealthe fluid reservoir. In a specific application, the platform lid 115 canpermit the array of wells to be observed (e.g., via the imagingsubsystem 194, via the naked eye) with the reservoir lid 164 in eitheran open or a closed position above the array of wells. Furthermore, thebase surface of the substrate platform can include an opticallytransparent and/or high-conductivity material that functions as a regionof access to the array of wells 120 secured at the substrate platform toenable optical interrogation and/or thermal modulation of the substrate110. In a preferred application, the substrate platform 105 can be usedto precisely and reproducibly place the substrate on a heating element(e.g., a thermocycler surface) of the thermal control module 190, inorder to better ensure reliable and uniform exposure of the array ofwells to the heating/cooling source. However, the substrate can beconfigured in any other suitable manner to engage with any othersuitable component of the system.

1.2 System—Array of Wells

The array of wells (set of microwells, microwells, wells) 120 functionsto capture the set of cells in addressable, known locations such thatthe set of cells can be individually identified, processed, andanalyzed. As such, the array of wells 120 is preferably configured tofacilitate cell capture in at least one of a single-cell format andsingle-cluster (e.g., a cell-particle pair) format. However, the arrayof wells 120 can additionally or alternatively be configured to receiveany other suitable type of particle, in any other suitable format. Forinstance, the array of wells 120 can be configured (e.g., sized, shaped)to receive mammalian cells, embyros, microspheres, particles,cell-particle pairs, and cells conjugated to microspheres.

As shown in FIG. 1 and FIGS. 3A-3B, the array of wells 120 is preferablydefined at the upper broad surface 112 of the substrate 110, each well128 in the array of wells 120 including a base surface 124 definedwithin the substrate and proximal a second side (e.g., lower broadsurface 114), an open surface 122 directly opposing the base surface 124and proximal the upper broad surface, and a set of walls 126 extendingbetween the base surface and the open surface defining the well cavity128 of the well.

The array of wells 120 is defined at an active region 116 of thesubstrate 110, wherein the active region can be any suitable area (e.g.,1 square inch, 10 cm, 2 square inch, 3 square inch, 4 square inch, etc.)of the substrate (FIG. 2A-2C). Preferably, the active region (and thearray of wells) of the substrate is accessible by other components ofthe system 100, including the imaging subsystem 194, fluid deliverymodule 140, thermal control module 190, and/or extraction module, inorder to perform isolation, processing, and analysis of single capturedcells. The array of wells 120 can include any suitable number of wells(e.g., on the scale of 10, 1,000, 10,000 wells, 50,000 wells, 100,000wells, 1 million wells, 2 million wells, 3 million wells, 4 millionwells, 5 million wells, 6 million wells, 7 million wells, 9 millionwells, 10 million wells, etc.). In preferred variations, the array ofwells includes at least 250,000 wells. In a specific example, the arrayof wells includes approximately 1 million wells (FIGS. 28A-28C and FIG.29). However, the array of wells can be configured in any other suitablemanner.

The open surface 122 is preferably an opening in the substrate 110 thatprovides access to the base surface 124 of a well 128, and is configuredto receive one of a single cell, a single particle, and a single clusterof cells or particles (e.g. a cell-particle pair), from a directionperpendicular to the upper broad surface 112 of the substrate 110. Forvariations in which the system is configured to retain a cell-particlepair, as shown in FIG. 3A, each of the cell and particle of thecell-particle pair can be received in sequence or simultaneously. Assuch, the open surface 122 can have a characteristic dimension (e.g.,width, diameter, circumference, etc.) that is larger than, smaller than,or equal to that of the base surface 124. In an example for capture ofcirculating tumor cells (CTCs) and a particle in single cell-particlepair format, the characteristic dimension of either the base surface 124or the open surface 122 can range between 20 to 40 micrometers, and theheight of the well cavity can range between 20 to 75 micrometers. Inanother variation, wherein the system is configured to retain either asingle cell or a single particle, as shown in FIG. 3B, thecharacteristic dimension of the base surface and/or the open surface canrange between 20 to 40 micrometers, and the height of the well cavitycan range between 10 to 40 micrometers. However, in other variations,any dimension of the wells within the array of wells, including wellcavity height and well cavity width, can be any value between 0.5microns to 50 microns, and can optionally be selected based on the assayto be performed by the system 100, the dimensions of the target cells,and/or the dimensions of the particles used. The open area of the arrayof wells 120 (i.e., the sum total area of the open surface of each wellin the set of wells) is preferably greater than 50% of the total area ofthe region of the substrate at which the wells are defined; morepreferably, the open area is greater than 80% of the total area. Howeverthe open area can be any suitable fractional area or percentage of thetotal area of the substrate.

The open surfaces of each well are preferably aligned flush with theupper surface of the substrate (e.g., at a surface plane 118), but canalternatively be slightly recessed within the substrate or otherwiseconfigured. Preferably, as shown in FIGS. 3A and 3B, the open surfacesof the wells of the array of wells are aligned with a surface plane 118of the substrate, wherein the horizontal axes of the open surfaces arecoaxial with the surface plane 118. In an example, the surface plane 118can be a plane with a lateral face parallel to the upper broad surfaceof the substrate, and defined at the intersection of the upper broadsurface of the substrate and a region of space superior the upper broadsurface of the substrate. In a specific example, the surface plane 118is a spatial boundary arranged between the upper broad surface of thesubstrate and a lower region of a fluid reservoir located superior thearray of wells, and defined at the interface between the open surfacesof the array of wells and a fluid path within the fluid reservoir. In apreferred application, cells and/or particles that are received into awell below the surface plane 118 are not accessible by fluid flow at theopen surface of the well, and are thus considered fully retained by thewell cavity 128 of the well, while cells and/or particles traversing thesurface plane 118 or remain above the surface plane 118 are accessibleby fluid flow and are transmitted downstream of the fluid path, and arethus considered partially and/or non-retained by the well cavity 128.However, the surface plane 118 can additionally and or alternatively bearranged with respect to any dimension of the substrate and/or the arrayof wells. Furthermore, the open surfaces of each well can be positionedwith respect to any region of the substrate, fluid reservoir, and/orfluid path.

In preferred variations, the open surfaces of each well are directlyfluidly coupled to a fluid path directly above and laterally superior tothe array of wells. To enhance fluid flow across the open surfaces ofthe array of wells, the open surfaces of each well can optionallyinclude a coating (e.g., hydrophobic, hydrophilic, electrostaticmaterial, chemoattractive, etc.) or physical features (e.g., texturized,notched, ridged, etc.). Furthermore, the open surfaces of each well canoptionally include passive or active retention features to retain andhold a single cell, or single cell-particle pair (e.g., physically orchemically triggered to increase or decrease open surface of well whenwell cavity 128 is occupied). In one example wherein the open surface122 has a characteristic dimension smaller than that of the base surface124, as shown in FIG. 4A, a well 128 can have a lip that forms aboundary of the open surface 122 in order to provide a characteristicdimension that is smaller than that of the base surface 124. The lip canbe planar or non-planar, and can further facilitate retention of asingle cell or a single cluster of cells at the well 128. FIGS. 4B to 4Edepict variations of the open surfaces of each well, which can defineany geometry for receiving a cell and/or particle into the well cavity,including a circular opening, rectangular opening, hexagonal opening, orany other suitable shape. The open surface 122 can, however, include anyother suitable feature that facilitates fluid flow, cell reception,and/or particle retrieval from the well 128 of the array of wells 120.

The base surface 124 is preferably parallel to, symmetrical to, anddirectly opposing the open surface 122; however, in some variations, thebase surface 124 can alternatively be non-parallel to, non-symmetricalto, and/or offset from the open surface 122. Similar to the upper broadsurface 112 of the substrate 110, the base surface 124 can be a planarsurface or a non-planar surface, and in variations of the base surface124 having a non-planar surface, the non-planar surface can includeconvex and/or concave portions having any suitable geometriccharacteristic, as shown in FIG. 5A. Additionally or alternatively, asshown in FIGS. 5B and 5C, the base surface 124 can be any one or moreof: textured (e.g., to facilitate desired fluid flow behavior, toattract or repel a given particle type, etc.), characterized by adesired porosity, characterized by a desired surface treatment,characterized by immobilized particles or biochemical moieties, andcharacterized by any other suitable feature that facilitates cellreception and/or retention in any other suitable manner. Though inpreferred variations, the base surface is closed such that there is nofluid flowthrough from the open surface of the chamber through thebottom surface of the chamber, the base surface can be alternativelyconfigured to include one or more fluid channels to allow egress ofparticles with characteristic dimensions less than the target cell inorder to exit the well cavity 128. However, the base surface can beotherwise configured in any other suitable manner.

In relation to the base surface 124 and the open surface 122, each well128 preferably has at least one wall (e.g., a set of walls) 126extending between the base surface 124 and the open surface 122. In avariation, as shown in at least FIG. 1 and FIG. 4A, the walls of eachwell 126 at least partially physically and fluidly separates anindividual well 128 from at least one other adjacent well, defines adepth, width, and/or cross-sectional dimensions of the well, and arepreferably perpendicular to a plane defined by the horizontal axis ofthe open surface 122. Preferably, the wall thickness of the walls 126 isbetween 4-5 micrometers, but can be any dimension less than 10micrometers. The wall 126 can extend vertically from a plane defined bythe open surface 122 to the base surface 124 to define the well cavity128; as such, in some variations, a well cavity 128 of each well in thethe array of wells can be prismatic (e.g., cylindrical prismatic,hexagonal prismatic, polygonal prismatic, non-polygonal prismatic,etc.). In a specific example, the well cavity of each well defines ahexagonal prism. However, as shown in the variations depicted in FIGS.6A and 6B, the wall 126 can extend between the open surface 122 and thebase surface 124 in any other suitable manner in other variations (e.g.,curved walls, straight walls, bent walls, etc.). For instance, the wall126 can gradually reduce a characteristic dimension (e.g., diameter,horizontal cross section, vertical cross section) of the well from theopen surface to the base surface (e.g., by forming discrete steps, bygradually adjusting the characteristic dimension in a linear or anon-linear manner with any suitable slope, etc.). However, in somevariations, a well 128 may not have a well-defined wall 126perpendicular to a plane defined by the open surface 122 (e.g., the basesurface may extend in some manner directly to the open surface withoutforming a wall perpendicular to the open surface). In examples, the basesurface 124 and the open surface 122 can be separated, with or without awall, by a distance (e.g., height of a well cavity 128) of between 0.5microns to 50 microns (e.g., approximately 25 microns for an applicationinvolving capture of single CTCs, approximately 40 microns for anapplication involving capture of single cell-particle pairs). However,the wells of the array of wells can be configured with any otherphysical characteristic and/or dimension, in order to perform theisolation, processing, and analysis steps described in method 200. In apreferred application, method 200 can include selecting an array ofwells with specific dimensions, numerosity, geometry, spatialarrangement and/or any other suitable characteristic, according to thedimensions of target cells desired to be captured, dimensions ofnon-cell particles utilized, and other parameters required to perform aspecific assay using system 100 (as described in Block S218).Additionally or alternatively, the set of walls can include a set ofchannels that fluidly couple each well to at least one adjacent well inthe array of wells 120. In such variations, the channel(s) of a set ofchannels can be defined within a region of the substrate 110 betweenadjacent wells, or can be defined by overlapping portions of adjacentwells. In a specific example, a channel can have a characteristicdimension of 5 microns, and in variations of the specific example, achannel can have a characteristic dimension ranging from 0.5 microns to75 microns.

The walls of the array of wells are preferably constructed from the samematerial as that of the substrate (as described in a previous section),but can alternatively be constructed of any other suitable material toconfer desired physical or chemical properties to the well cavities ofthe array of wells. For example, the walls can be configured to benon-permeable or semipermeable to various particles or fluids insolution that has entered the well cavities, and additionally oralternatively configured to be permanently or non-permanently rigid,flexible, or shape-changing (e.g., ability to expand open or collapseclosed) to control cell and/or particle entry into the well. In anembodiment of method 200, wherein the system 100 is used to capturesingle cell-particle pairs, wherein the cells are captured in a firststep and the particles are captured in a second step following the firststep, at least a portion of the walls of the each well in the array ofwells can be made of a shape-memory polymer, operable between a firstopen state and a second closed state. In an example, if a target cell iscaptured within a well at the first step, the walls of the well cavity128 can maintain the first open state to permit the capture of aparticle into the well at the second step, but if a target cell is notcaptured within a well at the first step, the walls of the well cavity128 can be activated to transition into the second closed state,essentially closing the open surface of each unoccupied well, which canincrease the efficiency of generating cell-particle pairs within thewells, and can help identify the wells of the array occupied by desiredtarget cells. However, the physical and chemical properties of the wellscan be configured in any other suitable manner to enhance theperformance of the system for any suitable application and/or variationof method 200 described in Section 2.

The internal surfaces of the well cavity 128 of each well in the arrayof wells (e.g., the sidewalls of the walls facing the interior of thewell cavity 128) can optionally be configured to interact with thecontents retained within the well cavity 128 (e.g., a captured cell,biological material, non-biological material, non-cell particles,cell-particle pairs, etc.). To permit such interaction, the internalsurfaces can include a functional feature (physical or chemical) orsurface-bound moiety on all sidewalls of the well cavity 128, but canalternatively be localized to any suitable portion or specific region ofthe well cavity 128 (e.g., at the base surface, proximal the opensurface, along the sidewalls, etc.). In a first variation, as shown inFIG. 7, the internal surfaces include a functional surface coating 131configured to bind to nucleic acid content that has been released from alysed captured cell, a probe or set of probes 36 released from anon-cell particle, and/or any other suitable captured entity within thewell. The functional surface coating 131 can be of synthetic, animalderived, human derived, or plant derived proteins that can bind to afunctional linker of a nucleic acid probe 36, as further described inSection 2. In an example, the functional surface coating permitsbiotinylated surface chemistry (e.g., biotin-streptavidin linkers) tobind to a probe including a functional linker. However, the functionalsurface coating of the internal surface of a well can be configured tobind to any contents retained within the well cavity 128 in any othersuitable manner. In a second variation, the functional surface coatingsare configured to physically retain and/or manipulate the capturedcontent, such as orienting a captured target cell or particle in aparticular direction for downstream analysis (e.g., optical imaging). Inan example, the functional surface coatings include a polymer or proteinproviding a sticky layer to adhere to a region of the captured targetcell (e.g., polymer adhesive, catechol-polystyrene, poly-D-lysine,fibronectin, collagen, vitronectin, etc.). In another example, thefunctional surface coating includes a polymer or protein that attracts acell towards the open surface of the well, or is a semi-permanentbarrier at the open surface of the well to control entrance of particlesinto the well. In a third variation, the internal surfaces of the wellare configured to add a chemical agent (e.g., a drug interacting withthe cell, an agent that controls pH of the solution within the well, anagent that controls density of fluid within the well, etc.), biochemicalagent (e.g., a fluorescent marker, antibodies, etc.), and/or a processreagent (e.g., a lysis buffer contained in a timed-released deliveryvehicle/microsphere, etc.), in order to perform downstream assays andanalysis of the captured cells. In a fourth variation, the internalsurfaces of the well can include physical features to increase,decrease, or vary the surface area (e.g., ridges, protrusions, pores,indentations within the well cavity 128). Furthermore, the physicalfeatures can include functionalized microparticles that have beenimmobilized within the well, reflective components to enhance opticalaccess and optical interrogation of contents retained within the well,and/or magnetic elements to manipulate the position of the cell orparticle within the well.

While every well 128 in the array of wells 120 can be substantiallyidentical, the array of wells 120 can alternatively include wells thatare non-identical to each other by any suitable feature (e.g.,morphological feature, mechanical feature, surface coating feature,thermal conductivity feature, electrical conductivity feature, etc.). Assuch, some variations of the system 100 can be configured to capture atleast one of multiple particle types and particles in multiple types offormats, in addressable locations, for processing and analysis. In afirst example, the array of wells 120 can include a first subset ofwells with wells having a first characteristic dimension (e.g., welldiameter, well depth, well volume, etc.) in order to capture a firstcell type in single cell format, and a second subset with wells having asecond characteristic dimension (e.g., well diameter) in order tocapture a second cell type in single cell format. In the first example,the first subset can be centrally located within the array of wells 120,and the second subset can be peripherally located within the array ofwells 120 and have a second characteristic dimension that is smallerthan the first characteristic dimension, in order to facilitate captureof larger particles at a central portion of the array of wells 120 andsmaller particles at a peripheral portion of the array 100 (e.g., in acytospin application). In one variation of the first example, the arrayof wells 120 can include wells having a gradient of characteristicdimensions in a radial direction (e.g., larger well dimensions towardthe center of the array and smaller well dimensions toward the peripheryof the array). In other variations of the first example, the array ofwells 120 can include wells having a gradient of any other suitablefeature characteristic (e.g., morphological feature, mechanical feature,surface coating feature, thermal conductivity feature, electricalconductivity feature, etc.) in a radial direction. In other examples,the array of wells 120 can include wells having a distribution (e.g.,gradient) of any suitable feature characteristic (e.g., morphologicalfeature, mechanical feature, surface coating feature, thermalconductivity feature, electrical conductivity feature, etc.) along anysuitable direction (e.g., linear direction, radial direction,circumferential direction, etc.).

In variations including subsets of wells, the subsets can be separatedfrom one another. In a first variation, each subset can be separatedfrom other subsets by a portion of the substrate in which no wells aredefined (e.g., a flat region of the broad surface). In a secondvariation, the subsets can be fluidically-isolated regions of acontiguous arrangement of wells, in which none of the wells of aparticular subset are fluidly coupled to a well of another subset. In aspecific example, the substrate defines twelve distinct subsets of thearray of wells 120, arranged in a two-by-six grid, that are separatedfrom adjacent subsets by flat region of the broad surface, with auniform spacing (e.g., 1 mm, 100 microns, 3 mm, etc.) between arrayedges. The subsets of wells can be further divided into groups (e.g.,groups of seven wells within a subset of 20,000 wells of a 250,000 wellset of wells), and any suitable interconnectivity between wells (e.g.,among subsets, between groups, etc.) can be provided by the set ofchannels of each well. Such configurations may permit efficient cellcapture (e.g., by a group including seven interconnected wells) bygroups of wells, while allowing the set of wells to be exposed tomultiple distinct samples (e.g., one sample per subset of the set ofwells). In an example, wells can be approximately 30 microns indiameter, 30 microns deep, and wall thicknesses of 4-5 microns (e.g.,which provides more efficient cell capture). However, in relatedvariations, the array of wells 120 can alternatively be subdividedand/or interconnected in any suitable manner. The subsets and/or groupsof wells can be arranged in any suitable manner. For example, thesubsets can be arranged in a rectilinear fashion (e.g, a grid layout ofwell subsets) and the groups can be arranged in a packed configuration(e.g., hexagonal close-packed, square lattice, etc.), and vice versa;the arrangement of the groups and subsets are preferably independent ofone another, but can alternatively be based on one another (e.g., thesubsets are arranged in a rectilinear fashion because the groups arearranged in a rectilinear fashion). Furthermore, each substrate 110 ofthe system 100 can have a single array of wells 120, or can havemultiple subsets of wells defined at the substrate in any suitablemanner (e.g., in a radial configuration, in a rectangular configuration,in a linear configuration, in a curvilinear configuration, in a randomconfiguration, etc.).

In a specific example of the array of wells, as shown in FIG. 8A, anarray of 250,000 wells in 144 mm² can be embossed into a plastic (e.g.,material COP480R) using a photolithographic etching process. A fluidreservoir 160 can then be provided (e.g., glued or otherwise attached)around the active region containing the array of wells that allows arelatively large liquid sample to be placed during use (e.g., 0.5 mL to5 mL). The specific example can further include two microchannels thatserve as inlet and outlet to the reservoir fluidly coupled to the arrayof wells at the active region. Furthermore, as shown in FIG. 8B, acell-containing sample, (e.g., up to 1 ml in volume), can be dispensedinto the fluid reservoir 160 formed by the recessed region of a firstplate 150 of a fluid delivery model surrounding the active region of thesubstrate. Cells present in the sample will settle down (e.g.,gravity-induced entry) over time through the fluid layer in thereservoir, and into the interior of the well cavity 128 through the opensurfaces of the wells. In specific applications, the settling timedepends on the size of the cells; typical cancer cells that are 10-25microns in size will settle in about 30 minutes. Once the cells enterthe well cavities, such that the entire volume of the cells is fullycontained within the well cavity 128 (e.g., fully retained, descendsbelow the surface plane 118, descends below the open surface of thewell), they are captured in single cell format. Because the walls inbetween each of the wells in the array of wells are thin (e.g., lessthan 10 microns thick, less than 5 microns thick, etc.), most of thecells tend to settle inside and fully retained within the well asopposed to on top of or partially retained by the wells. In specificapplications with cell-tracker stained cancer cells (SKBR3) spiked in 1ml PBS, the system 100 demonstrated an over 90% capture efficiency.

Furthermore, the array of wells 120 is preferably arranged in a packedarray, but can alternatively be arranged in any other suitable manner.In one example, the array of wells 120 can be arranged in a hexagonalclose-packed array, as shown in FIG. 9A. In another example, the arrayof wells can be arranged in a rectangular array, as shown in FIG. 9B. Inanother example, the array of wells 120 can be arranged in any suitableirregular or non-uniform manner, for instance, to facilitate fluid flowfrom one portion of the array of wells 120 to another portion of thearray of wells 120. In a specific example, the shortest distance of thecenter of each well to the center of an adjacent well of the array ofwells is approximately 30 micron. However, the array of wells 120 canalternatively be arranged with any suitable spacing between wells (e.g.,in a packed or a non-packed configuration), and in any other suitablemanner.

In a specific example configuration of the set of wells as shown inFIGURE o0, the array of wells is arranged in a hexagonal close-packedconfiguration, wherein each well of the array of wells includes ahexagonal open surface aligned with the broad surface (e.g., surfaceplane 118) of the substrate. Furthermore, each well includes a hexagonalfootprint at the base surface opposing the hexagonal open surface. Eachwell of the array of wells has a well cavity 128 that forming ahexagonal prism, including a set of walls approximately 5 micron inthickness, a height of approximately 40 micrometers, and acharacteristic width of approximately 25 micrometers. The substratedefines 267,000 such hexagonal wells within an active region of thesubstrate that is approximately 150 square millimeters. However, thearray of wells can be configured in any other suitable manner.

In some variations of the system 100, one or more wells of the array ofwells 120 can further include any other suitable element thatfacilitates stimulation and/or detection of a parameter (e.g., acellular response parameter) at the well(s) of the array of wells 120.In one example, one or more wells of the array of wells 120 of the arrayof wells 120 can include an electrode embedded in the substrate 110 at asurface of the well 128 in order to facilitate detection ofbioelectrical signals from contents of the well 128, and/or tofacilitate stimulation of the contents of the well 128. In variations ofthe example, the electrode can be embedded with an exposed portion atleast one of the base surface 124 and a wall 126 of the well 128. Inother examples, the well(s) can be coupled to channels that facilitatedelivery of process reagents to a cell/cell cluster at a well 128, orfacilitate extraction of contents of a well 128 (e.g., processedintracellular contents) from the well 128. The system 100 can, however,include any other suitable element that facilitates processing and/oranalysis of cells in at least one of single-cell format and singlecluster format.

1.3 System—Fluid Delivery Module

The system 100 can include a fluid delivery module 140 that functions totransfer a sample containing the population of cells, population ofparticles, and/or another fluid, such as a process reagent and/ordistribution fluid, to the array of wells 120, and can be coupled to thesubstrate. As such, the fluid delivery module can include an inlet 142,an outlet 144 and fluidic guides and/or structures that enable fluidtransfer into, out of, and throughout various portions of the system. Asshown in at least FIGS. 11A-11B, FIGS. 28A-28C, and FIG. 29, the fluiddelivery module 140 can include a first plate 150 arranged proximal theupper broad surface of the substrate 112, a second plate 156 arrangedproximal the lower broad surface of the substrate 114, and optionally, aclamping module configured to couple the first plate 150 to the secondplate, thereby positioning and/or aligning the substrate 110 between thefirst plate 150 and the second plate. Alternatively, however, the firstplate 150 can be directly coupled to the substrate 110 and/or to anyother suitable element of the system 100, such that the fluid deliverymodule 140 omits a second plate. As such, the fluid delivery module 140facilitates positioning of the substrate 110 to receive and/or seal thesample or fluid at the array of wells 120 (e.g., with a compressiveforce, with a hermetic seal, etc.). Additionally or alternatively, thefluid delivery module 140 can include a fluid reservoir 160 definedbetween the first plate and the broad surface of the substrate, andproviding a region for a fluid path 162 that facilitates controlledfluid flow through the reservoir across the array of wells 120.

In variations, the first plate 150 can have a rectangular footprint thatspans the upper broad surface 112 of the substrate 110. However, thefirst plate 150 can alternatively have any other suitable footprint(e.g., non-rectangular footprint, circular footprint, ellipsoidalfootprint, etc.) configured to span all or a portion of the upper broadsurface 112 of the substrate 110. In preferred variations, as shown inFIGS. 28A-28C and FIG. 29, the first plate 150 includes an opening orrecess 152 to be positioned over the array of wells 120. When the firstplate 150 is attached to the substrate 110, the recess 152 of the firstplate 150 defines the fluid reservoir 160 against the array of wells.The fluid reservoir 160 can be sealed by a reservoir lid 164, andadditionally and/or alternatively be placed within a substrate platformlid 115 (as shown in FIGS. 30A-30B, and FIG. 33), that can optionallyinclude an elastomeric gasket and a detent plunger to hermetically sealthe reservoir lid into the fluid reservoir. In another variation shownin FIGS. 11A and 11B, the first plate 150 includes a recess 152 at oneside of a closed surface of the first plate and facing the upper broadsurface 112 of the substrate 110, such that the recess 152 and the upperbroad surface 112 cooperatively define a lumen that can be fluidlyconnected to an inlet 142 and outlet 144 of the fluid delivery module.The lumen of the recess 152 preferably functions as a fluid reservoir160 to temporarily hold a sample and/or a processing reagent proximal tothe array of wells 120 (e.g., in a fluid layer and/or fluid path 162occupying the lumen defined by the recess and the broad surface of thesubstrate). As such, the recess 152 preferably spans the active regionof the substrate at which the array of wells 120 is defined, and alignswith the array when the first plate 150 is coupled to the substrate 110.The lumen (e.g., fluid reservoir) can have any suitable volume,preferably defined by the product of the gap distance between the basesurface of the recess and the projected area of the recess. The gapdistance (e.g., height of the fluid reservoir) is preferably between 25microns and 5 mm, but can alternatively be any suitable distance.

In one variation, the recess 152 can be a rectangular recess definedwithin the surface of the first plate 150 facing the substrate 110.Furthermore, the recess can have a substantially planar base surface, asshown in FIGS. 11A and 11B, or any other suitable base surface (e.g.,non-planar base surface). However, the recess 152 can alternatively haveany other suitable morphology. Additionally or alternatively, the recess152 can include a sealing element 157 (e.g., o-ring, sealant, etc.)surrounding a region of the recess 152 proximal the substrate 110, inorder to provide a hermetic seal upon coupling of the first plate 150 tothe substrate 110. Additionally or alternatively, the sealing elementcan be located on the substrate platform lid 115 and secured to thefirst plate in a closed configuration (FIG. 33). However, the firstplate 150 can alternatively be configured in any other suitable manner.

The second plate is configured proximal to a surface of the substrate110, directly opposing the broad surface of the substrate 110, andfunctions to provide a base to which the first plate 150 can be coupled,thereby positioning the substrate 110 between the first plate 150 andthe second plate. The second plate preferably provides a complementarysurface to which the surface of the substrate 110, opposing the upperbroad surface 112, can be coupled. In one variation, the second plate isa substantially planar, in order to provide a surface to which a planarsurface of the substrate 110 (e.g., a planar surface directly opposingthe broad surface of the substrate) can be coupled; however, the secondplate can be configured relative to the substrate 110 in any othersuitable manner. Furthermore, the second plate can include an aligningelement that facilitates alignment of the second plate relative to thesubstrate 110 and/or to the first plate 150. In variations, the aligningelement can include any one or more of: a protrusion and/or a recess atthe second plate that facilitates alignment, a track that facilitatesalignment, a magnetic element, and any other suitable alignment element.

In one variation, the first plate 150 is preferably coupled to thesecond plate with a coupling mechanism that can include one or more of:a pin, a screw, a magnetic coupler, a clamp, and any other suitablecoupling mechanism. To prevent obstruction, the coupling mechanism canbe located at peripheral portions of the system (e.g., at peripheralportions of the first plate 150, the second plate, and/or the substrate110), or at any other suitable location that does not interfere withfunction of the substrate. Alternatively, some variations of the system100 may omit the second plate, and have direct coupling between thefirst plate 150 and the substrate 110 in any suitable manner.

As shown in FIGS. 2A-2C and FIG. 14, some variations of the fluiddelivery module 140 can include a set of inlet and outlet channels(e.g., a set of fluidic pathways 146 associated with respective manifoldinlets 440 and manifold outlets 442) linking an inlet 142 of the system100 to each of the array of wells 120 and additionally or alternatively,the array of wells 120 to an outlet 144, wherein the outlet is fluidlycoupled to a receptacle for collecting removed fluids and/or samplefluid from the array of wells (e.g., to contain waste, to contain excessreagent, to collect desired sample for downstream processing). The setof fluid pathways 146 functions to distribute and route desired fluids(e.g., reagent-containing fluids, sample containing fluids, etc.) to thearray of wells 120 at substantially consistent fluid flow rates andvolumes. The set of fluid pathways 146 can have any suitablecorrespondence with the set of wells; for example, there may be onefluid pathway per single well 128, multiple fluid pathways 146 persingle well 128, and/or one fluid pathway connected to multiple wells.In another example, the set of fluid pathways 146 is a network of fluidpathways 146 that branches from a single fluid pathway, connected to theinlet 142, into a set of fluid pathways 146 connected to each wellindividually such that the total length of any fluid pathway between theinlet and a well is substantially equal in length (e.g., exactly equallength, equal to within 10-100 microns, equal to within a characteristiclength for a given flow rate and pathway cross-section, equal to withinany suitable threshold length, etc.). The set of fluid pathways 146 canadditionally or alternatively include fluid pathways 146 that connectgroups and/or subsets of wells to an inlet, as well as to other groupsand/or subsets of wells. Each of the subsets thus connected can includean identical number of wells, but can alternatively have differingnumbers of wells in each subset connected by the set of fluid pathways146.

In an example, the fluid delivery module comprises the first plate 150,and the first plate 150 defines an inlet 142 and an outlet 144. Theplate further defines a recessed region 152 that is fluidly connected tothe inlet, and that faces the broad surface of the substrate so as todefine a contiguous lumen (e.g., fluid reservoir 160) cooperatively withthe array of wells 120. The fluid delivery module is operable in a cellcapture mode, in which a fluid sample containing a population of cellsand/or a distribution fluid used to re-distribute deposited cells isflowed into the fluid reservoir 160 between the inlet and the outlet(e.g., by a pressure differential). In this example, the fluid sample isflowed substantially parallel to the broad surface of the substratethrough a fluid path 162. The sample is flowed at a flowrate (e.g., atleast 0.5 milliliters per second, 1 milliliter per second, 1 milliliterper minute, 1 microliters per second, 10 microliters per second, 100microliters per second, etc.), and the flowrate is selected (e.g.,controlled) such that the combination of vertical forces on the singlecells (e.g., gravitational, buoyancy, etc.) is directed toward the broadsurface, and is greater the lateral pressure forces from the surroundingfluid so as to promote settling of the single cells into the set ofwells from the laterally flowing sample.

The fluid reservoir 160 functions to receive a biological sampleincluding cells of interest and at least one fluid from the fluiddelivery module 140, and to deliver the biological sample and at leastone fluid to the set of fluid pathways 146 (e.g., of a manifold, aninlet manifold, an outlet manifold) to facilitate cell capture and/oranalysis. In a first variation, the fluid reservoir 160 includes anopening to atmospheric pressure, such that fluid delivery from the fluidreservoir 160 in an inlet-to-outlet direction is enabled by negativepressure applied by a pump in communication with a flow controlsubsystem 180 and coupled indirectly to the fluid reservoir 160 by atleast one of the set of fluid pathways 146 and the waste chamber. In thefirst variation, the negative pressure applied can be reversed in orderto facilitate flow in an outlet-to-inlet direction. In a secondvariation, the fluid reservoir 160 may not include an opening toatmospheric pressure, but can alternatively be coupled to a pumpconfigured to provide positive pressure and negative pressure at thefluid reservoir 160, in order to facilitate flow in both aninlet-to-outlet direction and an outlet-to-inlet direction,respectively. In a specific example of the second variation, the fluidreservoir 160 is coupled to a syringe pump configured to providepositive and negative pressure by manual pumping. Fluid delivery fromthe fluid reservoir 160 to the manifold can, however, be performed inany alternative suitable manner.

The fluid reservoir 160 can further comprise a level sensor, configuredto detect fluid level within the fluid reservoir 160, which functions toprevent gas bubbles from entering the set of fluid pathways 146. As suchthe level sensor can generate a signal upon detection of a trigger fluidlevel (e.g., a low fluid level as a threshold), and transmit the signalto a processor configured to receive the signal and generate a commandto control fluid delivery (e.g., via the flow control subsystem 180)into the set of fluid pathways 146 based upon the signal. The commandcan be used to automatically stop fluid flow from the fluid reservoir160 into the set of fluid pathways 146, and/or can function to implementcontrol of fluid flow in any other suitable manner. In variations of thefluid reservoir 160 comprising a level sensor, the level sensor can be aload cell, an ultrasonic level sensor, or any suitable signal configuredto generate a signal when fluid level in the fluid reservoir 160 passesa certain threshold. Detection of the signal can then generate aresponse to stop fluid flow within the system 100 and/or a response toadd more fluid to the fluid reservoir 160, thus preventing gas bubblesfrom entering the manifold. In a specific example, the fluid reservoir160 has a volumetric capacity greater than 6 mL, is configured to coupleto the manifold inlet 160 by a threaded male-female coupling, andcomprises an opening to atmospheric pressure, wherein the opening canalso couple to a syringe pump. In the specific example, the fluidreservoir 160 further comprises an ultrasonic level sensor configured togenerate a signal when fluid level in the fluid reservoir 160 passes acertain threshold. Other variations of the system 100 can altogetheromit the fluid reservoir 160 and use a network of fluid deliveryconduits, with or without valves, to deliver at least one fluid to theset of fluid pathways 146.

1.3.1. Fluid Delivery Module—Cartridge

The fluid delivery module 140 further functions to contain and deliverat least one fluid to the fluid reservoir 160, in order to facilitatecapture and/or analysis of cells within the array of wells. Preferably,as shown in FIGS. 12A and 12B, the fluid delivery module 140 comprises areagent cartridge 170 having a set of reagent chambers 172, each reagentchamber 176 in the set of reagent chambers configured to contain a fluidof a set of fluids to facilitate capture and/or analysis of cells. Thecartridge 170 can be cylindrical, conical, frustoconical, prismatic,pyramidal, or of any other suitable morphology. Each reagent chamber 176in the set of reagent chambers 172 is preferably identical to the otherchambers, but can alternatively be non-identical to other reagentchambers based on fluid storage requirements (e.g., volume requirements,temperature requirements, light exposure requirements, pressurerequirements). The set of fluids preferably comprises reagents includingbuffers (e.g., priming, wash, and permeabilization buffers), fixingsolutions (e.g., pre-fixing and post-fixing solutions), and cocktails(e.g., lysis, inhibitor, primary antibody, and secondary antibodycocktails), and can additionally or alternatively comprise stains (e.g.,fluorescent stains or histological stains) and any other suitable fluidsfor cell capture or analysis. In a first example, the set of fluids caninclude reagents used to perform on-chip cDNA synthesis from capturedmRNA, including lysis buffers, RNase inhibitors, and dNTPs. In a secondexample, the set of fluids can include reagents used to performexonuclease treatment of the contents within the array of wells toremove any single-stranded oligonucleotide sequence (e.g., from acaptured population of particles containing oligonucleotide probes). Ina third example, the set of fluids can include reagents for cDNAamplification using PCR master mix, dNTPs and primer sets. In a fourthexample, the set of fluids can include reagents for targetedamplification of products (e.g., genetic material, set of geneticcomplexes produced in variations of method 200) from the nucleic acidrecovered from single cells. In a fifth example, the set of fluids caninclude enzyme mixes and oligonucleotide sequences for ligating specificoligonucleotide sequences to single cell DNA or RNA. In a sixth example,the set of fluids can include enzymes mixes for tagmentation andlabeling of nucleic acids. In a seventh example, the set of fluids caninclude reagents that contain a population of SPRI beads used forsize-based purification and elution of nucleic acids of specific basepair lengths. However, the set of fluids can be otherwise configured andcan include any other suitable combination of reagents for any assaythat can be performed by system 100 and/or method 200. In variations ofthe system 100 configured to further promote purification of capturedcells by magnetic separation, the set of fluids can also comprisesolutions of magnetic beads coupled with affinity molecules configuredto bind to components of interest (e.g., undesired cells, fragments,waste products) within a biological sample. In one example, a reagentchamber 176 can contain a solution of streptavidin-coated magneticmicroparticles, configured to bind to CD45-bound white blood cells(WBCs). In alternative variations, the fluid delivery module 140 cancomprise a single chamber configured to facilitate delivery of a singlefluid or multiple fluids to facilitate capture and/or analysis of cellswithin a biological sample. In other variations, the chamber(s) of thefluid delivery module 140 can be replaced by any suitable fluidconduit(s).

The fluid delivery module 140 is preferably configured to be prepackagedwith at least one fluid (e.g., reagent, buffer, cocktail, stain,magnetic particle solution, etc.) inside a chamber, which functions tofacilitate capture and/or analysis of cells of interest according to aspecific, pre-defined protocol. Alternatively, the fluid delivery module140 can be prepackaged in an open or semi-open configuration, such thata user can transfer at least one fluid into at least one reagent chamber176 of the fluid delivery module 140 to facilitate capture and/oranalysis of cells of interest according to a different protocol.Preferably, at least part of the fluid delivery module 140 is configuredto be consumable, such that a portion of the fluid delivery module 140can be disposed of after one use or multiple uses. Alternatively, thefluid delivery module 140 can be configured to be reusable, such thatfluids can be repeatedly transferred to a reusable fluid delivery module140 configured to transfer fluids to the fluid reservoir 160.

In embodiments of the fluid delivery module 140 comprising a cartridge170 having a set of reagent chambers 172, each chamber is preferablyconfigured to be isolated from other reagent chambers and individuallyaccessible, which functions to control delivery of a specific fluid tothe fluid reservoir 160. In a first variation, the fluid delivery module140 comprises a set of reagent chambers 172, and comprises at least oneseal configured to seal the set of reagent chambers 172, thus isolatingeach chamber in the set of reagent chambers from other chambers. Theseal in the first variation is a puncturable foil seal, such thatpuncturing the seal at a chamber location provides access to the reagentchamber 176. In an example of the first variation, each chamber issealed at two locations and puncturing the seal at the two locationsexposes the chamber to atmospheric pressure, facilitating delivery of afluid within the chamber, through a location of puncture, to the fluidreservoir 160 by means of hydrostatic pressure. In another example ofthe first variation, each chamber is sealed and puncturing the seal at apuncture location, while providing a positive pressure at the puncturelocation (e.g., using a hypodermic needle, using a syringe pump, etc.)facilitates delivery of a fluid within the chamber to the fluidreservoir 160. In yet another example of the third variation, eachchamber is sealed and applying negative pressure at a chamber location(e.g., through a valve or an opening) facilitates delivery of a fluidwithin the chamber to the fluid reservoir 160. Puncturing a seal,applying positive pressure, and/or applying negative pressure at achamber can be performed manually, or can alternatively be performedautomatically using an actuation system configured to enable access tocontents of reagent chambers of the cartridge 170. The fluid deliverymodule 140 can alternatively facilitate individual access and/orisolation of a reagent chamber 176 using any other suitable mechanism orcombination of elements.

In a first specific example, as shown in FIGS. 12A and 12B, the fluiddelivery module 140′ comprises a substantially cylindrical cartridge 170comprising ten identical isolated reagent chambers 176, each configuredto contain a fluid or reagent to facilitate cell capture and/oranalysis. In the first specific example, the cylindrical cartridge 170can have one of an open configuration comprising open chambers, asemi-open configuration comprising open reagent chambers and sealedreagent chambers with prepackaged reagents, and a completely sealedconfiguration comprising sealed reagent chambers with prepackagedreagents. In semi-open or sealed configurations, sealed reagent chambersare sealed at two ends with a puncturable foil seal, and in open orsemi-open configurations, open reagent chambers are sealed at one endwith a puncturable foil seal. Each of the ten reagent chambers has avolumetric capacity of 4-6 mL and has a wedge-shaped cross section thatis substantially uniform along a majority of a 2″ length. In the firstspecific example, the cartridge 170 has a bevel at an inferior region ofthe cartridge 170, as shown in FIG. 12B, in order to facilitate fluidflow toward an inferior region of the cartridge 170, proximal the seal.

The fluid delivery module 140′ of the first specific example can also becoupled to an actuation system configured to individually access eachchamber of the cylindrical cartridge, in order to facilitate automaticdelivery of a fluid within each chamber to the fluid reservoir 160. Theactuation system of the first specific example comprises a rotary shaftdriven by a stepper motor, wherein the rotary shaft is mounted to thecylindrical cartridge. In the first specific example, the rotary shaftis mounted along an axis of rotation (e.g., a vertical axis of rotation)of the cartridge 170, such that the ten reagent chambers 172 surroundthe axis of rotation. This configuration, along with the stepper motor,functions to allow determination of the positions of the ten reagentchambers 172 as the cartridge 170 rotates during operation. Theactuation system of the first specific example also comprises a firstactuator configured to provide relative displacement between a firstpiercer and the cartridge 170, in order to facilitate piercing of a sealof a single reagent chamber 176 of the cartridge 170. In the firstspecific example, the first piercer is situated inferior to thecartridge 170, and comprises a puncturing tip, that aligns with reagentchambers 172 of the cartridge 170 in different rotational configurationsof the cartridge 170, wherein the puncturing tip is proximal to (e.g.,concentric with) and coupled to (e.g., contiguous with) a boundary of anaperture of the first piercer. As such, piercing of a seal of thecartridge 170 at a chamber location, by way of the puncturing tip,facilitates flow of contents of the chamber(s) through the aperture ofthe first piercer and into a fluid reservoir 160 configured to receivechamber contents. In some variations, the puncturing tip may also havean opening (e.g., an opening into a vertical channel, a slanted channel,or a channel with any other suitable orientation or path) to allow fluidto flow from the cartridge 170 to the fluid reservoir 160. Additionallyor alternatively, the structure of the puncturing tip can extend belowthe surface of the first piercer to allow fluid to drip in a guidedfashion toward the fluid reservoir 160.

In one variation of the first specific example, the actuation system candisplace the piercer relative to the cartridge 170 (e.g., in a verticaldirection, in a non-vertical inferior-superior direction) in order todrive the piercer into a seal of the cartridge 170. In this variation,the first piercer can be coupled to a drip plate that facilitates fluiddelivery into the fluid reservoir 160. In another variation of the firstspecific example, the actuation system can displace the cartridge 170relative to the piercer (e.g., in a vertical direction, in anon-vertical inferior-superior direction), in order to drive the seal ofthe cartridge toward the puncturing tip of the piercer. In still othervariations of the first specific example, the actuation system candisplace one or both of the cartridge 170 and the piercer in any othersuitable direction (e.g., vertical direction, a direction angularlydisplaced from a vertical direction, a horizontal direction) in order toenable piercing of a seal of the cartridge 170. As such, in somevariations of the first specific example, the cartridge 170 and/or thepiercer can be tilted away from a vertical or horizontal configuration.In tilted variations, fluid flow can be facilitated by gravity and/orapplication of positive or negative pressure to a reagent chamber 176 ofthe cartridge 170.

In a second specific example of the fluid delivery module 140″, theactuation system comprises a first actuator configured to drive a firstpiercer to puncture a seal at a first end of a reagent chamber 176, anda second actuator configured to drive a second piercer configured tocreate an opening in a second end of the reagent chamber 176. Puncturingthe first end of the reagent chamber 176 functions to vent the first endof the reagent chamber 176 to atmospheric pressure, in order tofacilitate fluid delivery from the reagent chamber 176, and creating anopening in a second end of the reagent chamber 176 functions to allowfluid within the reagent chamber 176 to flow from the reagent chamber176, to the fluid reservoir 160, due to hydrostatic pressure. In thesecond specific example, the first actuator is a solenoid actuatorconfigured to linearly displace the first piercer relative to a chamber,and to drive the first piercer into a puncturable foil seal at a firstend of the chamber. The second actuator is a rotary solenoid actuatorconfigured to convert rotary motion into linear motion, such that thesecond piercer coupled to the rotary solenoid actuator creates anopening in chamber through a puncturable foil seal at a second end ofthe reagent chamber 176. The first actuator and the second actuator,however, can be replaced or supplemented by any suitable actuator (e.g.,pneumatic or hydraulic actuator) or multiple actuators in variations ofthe second specific example. Furthermore, variations of the first andthe second specific examples can include any suitable actuator(s) thatenable a piercer to provide access to contents of a reagent chamber 176.Similarly, the stepper motor of the first and the second specificexamples can be replaced or supplemented by any suitable actuator orelement that enables determination of actuator position (e.g., anactuator coupled to a linear encoder). Thus, the actuation system of thefirst and the second specific examples facilitates rotation of thecartridge 170 to position individual reagent chambers 176 into alignmentwith at least one piercing element using a stepper motor, andfacilitates puncturing of individual reagent chambers using a subsystemof one or more actuators.

In both of the first and the second specific examples, the rotation ofthe cartridge positions a desired reagent chamber 176 directly intoalignment with (e.g., directly over) a fluid inlet 142 coupled to thefluid reservoir 160 and configured to receive and distribute contents ofthe reagent chamber 176 into a manifold (e.g., set of fluid pathways146), as shown in FIG. 13; however, in variations of first and thesecond specific examples, the cartridge 170, the reagent chamber 176,and/or the fluid reservoir 160 may be out of alignment (e.g., offset),but fluidly coupled in any suitable manner to facilitate fluid flow froma reagent chamber 176 to the fluid reservoir 160. In one example, asshown in FIG. 13B, the fluid reservoir 160 can be out of alignment withthe reagent chamber 176 of the cartridge, but coupled to a piercer (orthe reagent chamber 176) using a fluid conduit (e.g., a flexible fluidconduit). Furthermore, still other variations of the first and thesecond specific examples can omit rotation of a cartridge 170, or canadditionally or alternatively include translation of a cartridge (e.g.,in X, Y, and/or Z directions) to align desired cartridge reagentchambers 112 for delivery of processing fluids to a fluid reservoir 160.Furthermore, still other variations can include keeping the reagentcartridge fixed at a location (e.g., without rotating to access apiercer and/or inlet), and extracting fluid from the top of a desiredreagent chamber using a pipettor or capillary to aspirate and dispensefluid into the fluidic manifold, inlet, fluid reservoir, fluid pathways,and/or outlet of the array of wells as needed.

1.3.2 Fluid Delivery Module—Flow Control Subsystem

The system 100 can additionally include a flow control subsystem 180configured to control fluid and/or sample flow through the system 100,as well as reagent flow or the flow of any other suitable fluid throughthe system. The flow control subsystem is preferably operable in a flowmode, in which the flow control system applies a pressure gradientbetween the inlet and outlet of the fluid delivery module. The pressuregradient can be a positive pressure gradient (as defined between theinlet and outlet) or a negative pressure gradient, and it may be appliedcontinuously, periodically, asynchronously, in a reciprocating fashion(e.g., between positive and negative), or in any other suitable manner.In variations, the flow control subsystem comprises a pump configuredprovide at least one of positive pressure and negative pressure, andfunctions to facilitate fluid flow through the system 100. Preferably,the pump 182 is configured to provide both positive pressure andnegative pressure, such that fluid can flow in a forward direction andin a reverse direction within an element of the system 100. Flow in aforward direction preferably facilitates capture of cells of interestfrom a biological sample, and flow in a reverse direction preferablyfacilitates retrieval and/or analysis of cells of interest from thebiological sample. Preferably, the pump 182 is configured to couple tothe waste chamber and comprises a multi-way valve configured to providea connection at least between the pump 182 and the atmosphere, andbetween the pump 182 and the waste chamber. The pump 182, however, canadditionally or alternatively be coupled to any suitable element of thesystem to facilitate fluid flow, comprise a valve configured to provideany suitable alternative connection, and/or may not comprise a multi-wayvalve 162. In some variations, the pump 182 can also comprise a pressuresensor, which functions to enable measurement of a pressure provided bythe pump 182. In one example, the pump 182 is a syringe pump, however,the pump 182 can be any suitable pump configured to provide at least oneof a positive pressure and a negative pressure to facilitate fluid flowwithin the system 100. In order to minimize damage to cells, thepressures used for fluid delivery are low (e.g., less than 2 psi or lessthan 1 psi). In preferred variations, the pumping system can controlpumping pressures as low as 0.1 psi.

1.4 System—Thermal Control Module

The system 100 can additionally include thermal control module 190 thatfunctions to heat and/or cool the substrate and its contents, in orderto control the temperature of contents of the set of wells and/or fluiddelivery module during operation of the system 100. In variations, thethermal control module can heat and/or cool a biological samplecontaining cells of interest and/or a fluid to facilitate cell captureand analysis, and can further function to facilitate reactions requiringcycling from low to high temperatures, such as for cell lysis, enzymeactivations for probe hybridizations and thermocycling of biologicalsample mixtures for molecular diagnostic protocols, such as polymerasechain reaction (PCR). The thermal control module 190 can comprise aheater, a heat sink, a sensor, a fan, and one or more processors,however; the thermal control module can include any suitable componentto sense and modulate the temperature of the array of wells, andaccording to any instruction (e.g., user-input, automatically, pre-settemperature schedule, according to a particular assay, etc.). Invariations, the heater 192 of the thermal control module is preferably athin heater (e.g., Peltier device) configured to controllably heat andcool the biological sample and/or fluid. The thermal control module 190can additionally and or alternatively comprise a temperature sensor, orany other suitable element configured to facilitate temperature control.For example, the temperature sensor can couple to a heat-conductivesubstrate, to a heating element, or to a plate-shaped heater.Temperature control can be enabled using pulse-width modulation throughfuzzy logic control, a proportional-integral-differentiation algorithm,or any other suitable means. Temperature control can be provided to aresolution of 1° C., or any other suitable resolution given theapplication.

The heater 192 functions to modulate the temperature of the array ofwells 120 at the substrate 110, but can additionally or alternativelyinclude any suitable temperature control mechanism (e.g., anelectrothermal cooling plate). In variations, the heater can include alow-mass heater that interfaces with substrate 110 for thermocycling orincubation (e.g., of PCR components, reagents, and/or sample), and in aspecific example, the heater can comprise an aluminum heater coupled toa resistive power resistor (7 ohms) and a 2-wire 100-ohm RTD, whereinthe heater elements are connected to an in-house heater driver andtemperature controller. A PWM signal of 12 volts is provided across theheating element to heat the aluminum heater. The RTD providestemperature sensing and a control algorithm is used to modulate thetemperature. During cooling, heating is stopped and a fan is turned onto remove heat. Because the thermal mass is small, heating betweenanneal temperature (˜60° C.) and denaturation (˜94° C.) can be achievedin 20 seconds and cooling from 94° C. to 60° C. can be achieved in 40seconds with the specific example. In another specific example, thethermal control module can be used to maintain the temperature of thearray of wells below 10° C. (e.g., 5° C.) in order to preserve theviability of mRNA extracted from captured cells.

The heater is preferably a resistive electrothermal heating element, butcan alternatively or additionally include an induction heating element,convective heating element, optical heating element, or any othersuitable heating mechanism. Preferably, the heater is preferablyarranged adjacent to a bottom surface of the substrate, wherein thelateral broad face of the heating element is directly coupled to thelower broad face 114 of the substrate beneath the array of wells, butcan alternatively be positioned adjacent to a top surface of thesubstrate 112, distal the substrate (e.g., in variations wherein theheater includes non-contact heating mechanisms) at either the bottom ortop surface of the substrate, or in any other suitable location relativeto the substrate.

In a first variation, the heater 170 comprises a heat-conductivesubstrate coupled to a heating element. In the first variation, theheat-conductive substrate preferably houses the heating element;however, the heating element can alternatively be configured to contacta surface of the heat-conductive substrate. The heat-conductivesubstrate can be composed of a conductive material (e.g., silicon,aluminum, copper, gold, silver), or any other suitable material fortransferring heat from the heating element. Preferably, theheat-conductive substrate maintains temperature uniformity over aheating surface with less than 1° C. variability over the heatingsurface; however, the heat-conductive substrate can provide any suitabletemperature profile over a heating surface. In the first variation, theheat-conductive substrate preferably has a thin profile (e.g., has adimension less than 4 mm thick), to reduce the energy required to heatthe heat-conductive substrate to a specified temperature. Theheat-conductive substrate can be further configured to provide cooling.In a specific example of the first variation, less than 50 (e.g., 40,30, 20, 10) Watts of power is required to heat the heat-conductivesubstrate to a temperature of 100° C. from room temperature within anappropriate amount of time.

In an example of the first variation, heating through one face can beaccomplished by using a plate-shaped resistance heater that has oneexposed face and thermal insulation covering all other faces. In anotherexample of the second variation, heating can be provided through oneface of a heater by using a Peltier heater. In a variation of the heater192 using a Peltier heater, the heater 192 comprises a thermoelectricmaterial, and produces different temperatures on opposite faces of theheater 192 in response to a voltage difference placed across thethermoelectric material. Thus, when a current flows through the Peltierheater, one face of the Peltier heater lowers in temperature, andanother face of the Peltier heater increases in temperature. The system100, however, can further comprise any other suitable heater 170configured to heat a biological sample and/or a fluid.

The thermal control module can also include a second heating elementadditionally and/or alternatively to the heater 192 described above,that functions to modulate and control the temperature of fluidsprovided to the array of wells during reagent delivery. As shown inFIGS. 15A and 15B, a fluid heater 154 (e.g., an aluminum heater with adefined geometry) can be coupled within the recess 152 of the fluiddelivery module 140. The fluid heating plate 154 is preferably coupledto a plate (e.g., the first plate 150 of the fluid delivery module 140),wherein the first plate 150 is configured to facilitate coupling of theset of fluid pathways 146 (e.g., via the manifold) to the array ofwells. In one variation, the fluid heater 154 is coupled to a surface ofthe first plate 150, and in another variation, the fluid heater 154 isembedded within the first plate 150. The plate 150 is preferablyconfigured to facilitate heat transfer between the fluid heater 154 andthe fluid within the fluid reservoir coupled to the array of wells, suchthat a biological sample and/or a fluid within the array of wells can beappropriately heated. The plate 150 can be further configured to provideconductive cooling through a fluid path 162; however, cooling may not beprovided, or can be provided using any other suitable element (e.g., afan blower coupled to provide forced air cooling as necessary). Invariations wherein the plate 150 is configured to provide cooling,cooling can be enabled by flowing a coolant (e.g., water, oil, air,composite liquid) through a fluid path 162, and controlled using acontrolled fluid pump.

As such, a variation of the system 100 as shown in FIG. 16 can furthercomprise a fluid heater 154 coupled to the first plate of the fluiddelivery module within the recessed region, the fluid heater 154 atleast partially defining the fluid layer through which a convective flowprovided by the fluid heater 154 can flow in the second directionparallel to the broad surface, wherein, with the fluid heater 154, thesystem is operable in a diffusion mode that provides diffusion transportbetween the convective flow and the set of wells. In more detail, theopen surfaces of the set of wells are very small compared to the regionof the reservoir where convective reagent flow through the fluid path isestablished. Reagents can be transported from the fluid layer into theset of wells by diffusive transport. The time required for diffusion ofa reagent into a well (approximately 30-50 microns deep) can beestimated using the formula, Diffusion Time ˜(DiffusionLength)²/Diffusivity, and the timing, velocity, and temperature at whichmultiple and/or consecutive reagents are dispensed to the array of wellscan be appropriately adjusted and/or optionally automated to account forproper uniform exposure of the contents of the wells to the reagentsduring various biochemical assays. For example, a small molecule, suchas PCR primer (Diffusivity 10⁻⁶ cm²/s) would take approximately 9seconds to diffuse into the well. Taq Polymerase with diffusivity ofapprox. 4.7×10⁻⁷ cm²/s would need about 19 seconds to diffuse across thewell cavity. Thus, in order to deliver “all-in-one” (e.g., sequential,simultaneous, single, multiple, mixtures of reagents) PCR reagents intothe array of wells, reagents are convectively transported into the fluidreservoir given the provision of sufficient time (around 2-3 minutes)for the reagents to diffuse into the array of wells. In variations, fora well containing a cell-particle pair with a well cavity height ofapproximately 40 microns, the presence of an impermeable non-cellparticle on top of a cell can obstruct the diffusive fluid path betweenthe fluid path 162 within the fluid reservoir 160 and the well cavitiesbelow, thereby requiring at least two (e.g., three, four, five, ten)times more diffusion time for reagents to arrive at the captured cellthan for wells containing only a single cell.

1.5 System—Imaging Subsystem 194

The system 100 can additionally include an imaging subsystem 194 thatfunctions to image the contents of the set of wells, and can furtherfunction to distinguish target objects (e.g., CTCs, labeled cells,microspheres) captured in the set of wells from other cells or objectsin the sample introduced into the system 100. The imaging subsystem 194preferably includes a fluorescence microscope, but can additionally oralternatively include any suitable imaging mechanism (e.g., an opticalmicroscope, a CCD camera, a photodiode array, a light emitting diode,reflectors, one or more processors etc.). The fluorescence microscope ispreferably operable (e.g., in an identification mode, a detection mode,etc.) to detect a fluorescence signal emitted from a target object oneor more of the set of wells, and thereby identify that the well(s)contain(s) a target object. In a specific example, the imaging system(e.g., fluorescence imaging system) can be operable in a mode forproviding real-time or near real-time fluorescence imaging of samplesprocessed according to an assay. The imaging subsystem 194 is preferablypositioned beneath the substrate and oriented to image the contents ofthe set of wells through the transparent (or translucent) material ofthe substrate; alternatively, the imaging subsystem 194 can bepositioned above the substrate and oriented to image the contents of theset of wells unobstructed by the material of the substrate itself.However, the imaging subsystem 194 can be otherwise positioned in anysuitable manner.

Additionally or alternatively, the system 100 can include any othersuitable element that facilitates cell processing and/or analysis. Forinstance, the system 100 can include optical elements (e.g., embeddedwithin the substrate 110, coupled to the substrate 110) that function tofacilitate imaging. The optical elements function to adjust incominglight, preferably to facilitate imaging. The optical elements canfunction to bend, reflect, collimate, focus, reject, or otherwise adjustthe incoming light. The optical elements are preferably defined withinthe substrate 110, but can alternatively be defined by any othersuitable component of the system 100. Optical elements can include anyone or more of: light reflectors disposed within the substrate thicknessadjacent the array(s) 110 defined on a surface of the substrate 110opposite that defining the array of wells 120, microlenses defined on abroad surface of the substrate 110 proximal that defining the array ofwells 120, light collimators, light polarizers, interference filters,light reflectors (e.g., 900 illumination elements), elements thatminimize excitation rays from going into path of collected fluorescenceemission light, diffraction filters, light diffusers, and any othersuitable optical element. The system 100 can additionally oralternatively include well affinity mechanisms that function to attracta cell of interest 10 towards a well 128. Well affinity mechanisms caninclude electric field traps, affinity moieties (e.g., coated to a wellsurface), features (e.g., microfluidic features) that direct flow intoan element, or any other suitable pore affinity mechanism. The system100 can, however, include any other suitable element(s).

In a variation, as shown in FIG. 17, the imaging subsystem 194 caninclude an ultraviolet illumination element that functions to sterilizewells, and additionally or alternatively to irradiate wells containinglight-reactive components, such as photo-cleavable chemical bonds. Theultraviolet illumination element can include one or more light emittingdiodes, mercury vapour lamps, and/or metal halide lamps, that emitwavelengths between 300 to 400 nm. In a specific application, asdescribed in Section 2.4, UV illumination at ˜365 nm wavelength can beperformed for 5 to 20 minutes at the array of wells to separate a set ofprobes 36 from a particle, by cleaving a photo-sensitive bond couplingthe set of probes 36 to the particle (e.g., biotinylated chemistry). Insome variations, wherein the illumination element is positioned superiorthe array of wells, the heating element of the thermal control modulebelow the array of wells can include a reflective surface that canenhance the uniform illumination of the particles within each well inthe array of wells. However, photo-illumination of the contents of thearray of wells can be performed in any suitable manner, and by anyconfiguration n of the illumination element. Furthermore, the imagingsubsystem and/or any other component of system 100 can also includereflective surfaces positioned at different locations in relation to thearray of wells, in order to uniformly illuminate the interior of eachwell cavity. In one example further described in Section 2, the heatingsurface of the thermal control module 190 below the substrate can bereflective, thus permitting UV light to illuminate all surfaces of thecaptured particles for photocleaving molecules (e.g., set of probes,genetic complexes,) from the outer surfaces of the particles and/or theinterior surfaces of the well cavities. However, incident light from theimaging subsystem into the array of wells can be modified, reflected,refracted, diffused, and/or otherwise manipulated by any other featureof any other component in system 100 to improve optical interrogation ofthe contents of each well.

The imaging subsystem 194 can also further comprise a tag identifyingsystem comprising a detection module and at least one tag configured toprovide information. The tag identifying system functions to readbarcodes, QR codes and/or any other identifying tags of the system 100,and to communicate information from the identifying tags to a processor.The tag identifying system can be coupled to the illumination module110, to facilitate identification and reading of tags located on imagingsubstrates coupled to the platform, or any other suitable systemelement. In other variations, the tag identifying system may not becoupled to the illumination module. The tag identifying system ispreferably fixed in location, but can alternatively be configured tomove relative to other system elements. In one alternative variation,the tag identifying system can be a standalone unit that is configuredto be manipulated by a user to scan tags or labels located on elementsof the system 100. The tag identifying system can comprise a barcodereader, a radio-frequency identification (RFID) reader, a QR codereader, a nearfield communication device, or any other suitable elementimplementing a mechanism that can identify a unique identifier locatedon the an imaging substrate or other aspect of the system 100 (e.g.,glass slide, cartridge, array of wells, etc.). The tag identifyingsystem can alternatively or additionally be configured to parse andinterpret non-encoded information (e.g., text) on an identifying tag. Insome variations of the system 100, the optical sensor of the imagingsubsystem 194 can additionally function as a tag identifying system.

Preferably, a tag intended to be identified and/or read by the tagidentifying system preferably communicates information to the tagidentifying system upon being read. The information can compriseinformation related to imaging substrate (e.g., array of wells, glassslide) identification information, protocol information (e.g., stainingprotocol information), information related to suggested systemparameters required to actualize a protocol, information related tocalibration of the system 100 with regard to a specific imagingsubstrate, information related to contents of an imaging substrate,information configured to facilitate positive location identification ofan imaging substrate or locations within an imaging substrate, and/orany other suitable type of information. The information can be coupledto (e.g., embedded within) image data captured by the optical sensor,and/or can be communicated to the processor using any other suitablemeans.

1.6 System—Additional Elements

In embodiments of the system 100 configured to promote furtherpurification of captured cells, the system 100 can further comprise amagnet 90 that enables separation of captured cells from undesiredsample materials. The magnet 90 is preferably a single magnet, but canalternatively be one of multiple magnets (e.g., lined up in parallel),in order to provide a greater magnetic flux to capturemagnetically-bound particles. Preferably, the magnet or group of magnets90 is coupled to a magnet holder of the system 100, wherein the magnetholder is configured stabilize the position of the magnet(s) of thesystem 100 to provide experimental consistency. Additionally, the magnet90 is preferably configured to be positioned proximal to the fluidreservoir 160, such that purification of captured cells is facilitatedwithin the fluid reservoir 16 o by a magnetic field provided by themagnet; however, in alternative variations, the magnet can be unfixed orfixed relative to any suitable element of the system. In an example, themagnet 90 is a rectangular prism-shaped magnet 90 fixed to the manifold(e.g., set of fluid pathways 146) proximal to the fluid reservoir 160and contacting a wall of the fluid reservoir 160, such that particles ofa sample bound to magnetic beads can be reversibly captured at a wallwithin the fluid reservoir 160. In another example, the magnet can beconfigured to provide a magnetic field at the manifold, at the array ofwells 180, or at an outlet fluid reservoir 160, such thatmagnetically-bound particles can be captured within at least one of themanifold, the array of wells 180, and the outlet fluid reservoir 160during processing and/or purification.

The system 100 can further include an extraction module (e.g., cellretrieval subsystem) that functions to extract at least one of a singlecell and a cell cluster from a well 128 of the array. While anindividual cell from a single well 128 is preferably selectivelyremoved, the extraction module can facilitate simultaneous multiplecell/cell cluster removal from the array of wells 120. The cell/cellcluster is preferably removed by applying a removal force to the cell.The removal force is preferably applied by aspirating the contents outof a well 128 (i.e., using a negative pressure); however, the removalforce can additionally or alternatively be applied by pumping fluidthrough the array of wells 120 (e.g., by way of a perimeter channel 150)to provide a positive pressure that drives the cell/cell cluster fromthe well 128. In one variation, the pump pressure provided by a pumpmechanism at the extraction module is less than 10,000 Pa, and in aspecific variation, the provided pump pressure is 6,000 Pa. In anotherspecific variation, the provided pump pressure is 1,000 Pa. However, anyother suitable pump or aspiration pressure can be used.

In some variations, the extraction module can comprise a particleextractor. The particle extractor functions to selectively remove one ormore isolated cells and/or non-cell particles from an addressablelocation within the system 100. The particle extractor is preferablyconfigured to remove a cell/particle cluster from a single well 128, butcan alternatively be configured to simultaneously remove multiplecells/particle clusters from multiple wells. The extraction module ispreferably operable in an extraction mode, wherein in the extractionmode the extraction module extracts at least one of a set of singlecells and a set of non-cell particles from a well of the set of wells,along a direction normal to the base surface of the well. In theextraction mode, the fluid delivery module is preferably removed fromthe substrate; however, the fluid delivery module can alternativelyremain coupled to the substrate when the cell removal module is operatedin the extraction mode. The particle extractor can, however, compriseany other suitable cell removal tool such as that described in U.S.application Ser. No. 13/557,510, entitled “Cell Capture System andMethod of Use” and filed on 25 Jul. 2012, which is herein incorporatedin its entirety by this reference.

In a first variation of the particle extractor, the particle extractoris configured to access the array of wells 120 from a direction normalto the upper broad surface 112 of the substrate 110. The particleextractor preferably removes the cell/particle cluster in asubstantially normal direction from the upper broad surface 112 of thesubstrate 110, but can alternatively remove the cell/particle cluster inan angled direction relative to the upper broad surface 112 of thesubstrate 110. The particle extractor preferably includes a hollowchannel (e.g., of a micropipette, capillary tube, etc.) that accessesthe array of wells 120 and defines a substantially fluidly isolatedvolume in fluid communication with one or more wells. The hollow channelcan include one or more sealing elements at the tip (e.g., a polymericcoating or adequate geometry) that facilitate fluid seal formation withthe well(s). The particle extractor preferably tapers from a proximalend to the tip, in order to provide an adequate geometry to receivecontents of a well into the particle extractor; however, the particleextractor can alternatively have any other suitable form. As such, thehollow needle is preferably configured to form a substantially fluidlyisolated volume within a well 128 of interest, and a low-pressuregenerator (e.g., a pump) is then used to aspirate the retained cell/cellcluster out of the well 128, through the hollow channel, and into a cellcollection volume of the particle extractor. In one variation, theparticle extractor is a micropipette having a height of 200 micrometersand a hollow channel diameter of 25 micrometers; in another variation,the particle extractor is a capillary tube having a channel diameter of150 micrometers. In another variation, the wells of the array of wells120 are grouped such that each group may be circumscribed by a closedcurve in the plane parallel to the broad surface of the substrate, andthe particle extractor has an inner diameter that is smaller than thelargest chord of the closed curve. However, other variations of thesespecific examples can have any other suitable defining dimensions.

Cell and/or non-cell particle removal from the system 100 is preferablyautomated, but can additionally or alternatively be semi-automated ormanual. Furthermore, cell and/or non-cell particle removal can beperformed along with cell identification, comprising automatic fixing,permeabilization, staining, imaging, and identification of the cellsremoved from the array of wells 120 through image analysis (e.g.,through visual processing with a processor, by using a light detector,etc.) or in any other suitable manner. The extraction module can beconfigured to facilitate advancement of a particle extractor to a well128 containing a cell/particle cluster of interest, for instance, withan actuation subsystem. The extraction module can additionally oralternatively be configured to facilitate cell and/or particle removalmethod selection and/or cell removal tool selection. In anothervariation, cell identification at the extraction module can besemi-automated, and cell and/or particle retrieval can be automated. Forexample, cell staining and imaging can be done automatically, whereinidentification and selection of the cells of interest can be donemanually. In another variation, all steps can be performed manually.However, any combination of automated or manual steps can be used.

Variations of the system 100 can be operable to facilitate assays in amanner analogous to the methods described in U.S. application Ser. No.15/333,420 entitled “Cell Capture System and Method of Use” and filed 25Oct. 2016, U.S. application Ser. No. 14/163,185 entitled “System andMethod for Capturing and Analyzing Cells” and filed 24 Jan. 2014, U.S.application Ser. No. 14/863,191 entitled “System and Method forCapturing and Analyzing Cells” and filed 23 Sep. 2015, and U.S.application Ser. No. 14/289,155 entitled “System and Method forIsolating and Analyzing Cells” and filed 28 May 2014, which are eachincorporated in their entirety by this reference. The system isadditionally or alternatively operable for a variety of on-chip (e.g.,in situ at the substrate) analyses and assays, including: on-chipimmunochemistry, on-chip DNA and/or mRNA FISH, on-chip mRNA and/or DNAPCR, on-chip isothermal amplification, on-chip live cell assays, on-chipcell culture, and other similar assays.

In a specific example, the system may be operated according to thefollowing procedure: a 4 milliliter whole blood sample is partiallyfixed with an equal volume of 0.4% PFA for 10 minutes; the sample isenriched for cancer cells and washed with PBS; the cells, comprisingapproximately 85% cancer cells and approximately 20,000 whole bloodcells are backflowed with 1 milliliter PBS to generate a backflowsolution containing the cells; the backflow solution is flowed over thearray of wells 120 of the system 100 and the cells are captured by thewells; an immunostaining reagent is flowed by way of the fluid deliverymodule to each of the wells of the set of wells; cancer cells areidentified using a fluorescence microscope which detects a fluorescencesignal emitted by any cells which are successfully tagged by theimmunostaining reagent; identified cells are extracted from theircorresponding wells using the cell removal module of the system (e.g., acapillary tube on a three-axis traversing stage) and transferred to aPCR tube, where the single cell genome is amplified; the amplifiedsingle cell genome is packed for downstream processing (e.g., wholegenome sequencing, targeted sequencing, etc.).

Additionally, as a person skilled in the field of cell sorting willrecognize from the previous detailed description and from the figuresand claims, modifications and changes can be made to the embodiments,variations, examples, and specific applications of the system 100described above without departing from the scope of the system 100.

2. Method

As shown in FIG. 18, a method 200 for isolating and analyzing apopulation of target cells comprises: receiving a population of targetcells into an array of wells in Block S210; distributing a population ofparticles into the array of wells in Block S220, wherein each particleof the population of particles can optionally be coupled to a set ofprobes 36 having a binding affinity for a biomolecule associated withthe population of target cells; redistributing a subset of partiallyretained particles across the array of wells in Block S230; andprocessing the array of wells in Block S240. In some variations, BlockS240 can include: generating a set of genetic complexes 70 comprisingreleased nucleic acid content from the population of target cells andportions of the population of particles (e.g., the set of probes 36) inBlock S242, and additionally or alternatively performing a biochemicalprocess at the array of wells in Block S244. Furthermore, method 200 canadditionally or alternatively include removing the set of geneticcomplexes 70 generated in Block S240 from the array of wells fordownstream analysis in Block S250.

The method 200 functions to enable isolation, capture, and retention ofcells, more preferably to enable efficient capture of cells insingle-cell format and/or single-cluster format (e.g., a pair of asingle cell and a single particle colocalized within the same well), atknown, addressable locations, and further to facilitate performance ofmultiple single-cell/single cluster assays that can be performed onindividual cells or cell clusters (e.g., rare cells in a biologicalsample, a cell-particle pair). In a preferred embodiment, as shown inFIG. 19, the method 200 functions to achieve an ideal state for a subsetof the array of wells, wherein a well in the ideal state receives asingle cell in Block S210, followed by a single particle in Block S220,in order to capture individual cell-particle pairs in single-clusterformat within the array of wells. However in variations, the method 200can achieve the ideal state for a subset of the array of wells that havereceived more than a single cell and/or particle, by redistributing thepopulation of cells and/or particles that exceed the capacity of thewell (e.g., traversing a spatial boundary defined by the surface plane118 of the substrate), thereby correcting errors (e.g., aggregation,over-saturation) in the distribution steps in Block S210 and Block S230to improve the efficiency of single-cell and/or single-cluster capturewithin the array of wells. In additional variations, the method 200 canredistribute the population of cells and/or particles that have not yetentered the array of wells as a result of previous distribution steps(e.g., remaining above the surface plane 118), thereby permittinguncaptured cells and/or particles additional opportunities to bereceived into accessible wells, and increasing capture efficiency of theintroduced cell and/or particle populations. Specifically, throughimplementation of variations of the preferred embodiment, the steps ofmethod 200 can increase the capture efficiency of single cell-particlepairs to greater than 80% capture efficiency, as compared to less than20% capture efficiency by other methods. The steps of method 200 can beused to achieve the ideal state for a subset of the array of wells inany sequence or number of repetitions, and can additionally and/oralternatively be used to manipulate the contents of the array of wellsto hold any number of cells, non-cell particles, and/or any combinationthereof (e.g., combinations of one or more cells, one or more particles,etc.) in order to permit downstream analysis of the population ofcaptured cells.

In a preferred application, method 200 can function to enable downstreamprocessing of the array of wells for genetic analysis. In a variation,as shown in FIG. 20, a single cell-particle pair that has been capturedwithin a well can be processed within the well to form an identifiablegenetic sequence, wherein nucleic acid content of the cell can bind to acomplementary nucleotide probe coupled to the particle. In this way,method 200 can provide significant benefit to increasing the number ofcells that can be processed in a short amount of time by quicklyisolating and retaining cell-particle pairs, and performing biochemicalprocesses upon the cell-particle pairs without necessitating removalfrom the array of wells. In some embodiments, the method 200 can be usedto capture and facilitate analyses of circulating tumor cells (CTCs) andsubpopulations of CTCs, such as circulating stem cells (CSCs), but canadditionally or alternatively be used to capture any other suitable cellof possible interest for processing and analysis. However, method 200can be implemented in any other suitable manner for any other suitableapplication in which high-throughput cell/particle isolation or pairingis desired.

In variations of the method 200, Blocks S210, S220, and/or S230 can beperformed with any suitable number of repetitions, according toprotocols for processing the cell population according to differentassays. Furthermore, Blocks S210, S220, S230, and S240 can be performedin any suitable order or simultaneously, according to protocols forprocessing the cell population according to different assays. In analternative embodiment of method 200, distribution of particles in BlockS220, and additionally or alternatively re-distribution in Block S230,can be performed prior to distribution of target cells in Block S210.

In a variation of this alternative embodiment of method 200, as shown inFIG. 24, FIG. 25, and FIG. 26, Block S220 can be performed prior toBlock S210, which functions to enable single-particle capture of thepopulation of particles within each well of the array of wells, whereinthe population of particles can serve as delivery vehicles to deliver aset of labeled probes to each well. Preferably, each particle of thepopulation of particles is detachably coupled to a probe or set ofprobes 36 comprising a unit identifier common to all probes in the setof probes 36 (FIG. 7). Once a single particle has been successfullycaptured into a well, the set of probes 36 may be released from theparticle and can be bound to the interior surface of each well cavity.The particle can be optionally removed from the well, thereby enablingfacile access of single cells to the array of wells for single-cellcapture as described in Block S210. Once single cells have been capturedwithin the wells, wherein each well contains a set of probes 36 uniqueto the individual well via the unit identifier of the set of probes 36,biomolecules, including genetic content, of the captured cells can bereleased and bound to the sets of probes to generate genetic complexes,as described in variations of Block S240 (FIGS. 24, 25 and 26). Removal,processing, and/or analysis of the genetic complexes can be performed inBlock S250. As shown in FIG. 24 a method 200′ for isolating andanalyzing a population of target cells comprises: receiving a populationof particles into an array of wells in single-particle format, whereineach particle includes a set of probes 36; capturing a population oftarget cells into an array of wells in single-cell format, wherein eachcell can interact with a corresponding set of probes 36 within eachwell; receiving a process reagent into the array of wells to perform abiochemical process at the array of wells; and processing the product ofthe biochemical process from the array of wells. In some variations,method 200′ can include: binding the set of probes 36 of the populationof particles to the array of wells; generating a set of geneticcomplexes 70 comprising released nucleic acid content of each cell andthe set of probes 36 within each well; processing the genetic complexeswithin the array of wells; and removing the set of genetic complexes 70from the array of wells. The method 200′ can function to enable theisolation, capture, and labeling of single cells for generating geneticlibraries in single-cell format in a high-throughput manner, withoutnecessitating the need for additional removal steps from the array ofwells for downstream processing. By labeling each of the wells with aset of probes 36 comprising a unique label (particles captured insingle-cell format), cells captured in single-cell format can besubsequently processed using the corresponding set of probes 36localized within the same well. However, variations of steps of method200 can be performed in any suitable order to isolate and lable geneticmaterial originating from single cells, and achieve generation ofgenetic complexes that can be easily identified, processed, andanalyzed.

The method 200 is preferably implemented at least in part using thesystem 100 described in Section 1 above; however the method 200 canadditionally or alternatively be implemented using any other suitablesystem 100 for cell capture and analysis. As described in a variation ofSection 1 above, the well dimensions can be configured and selected toretain cells and/or particles below a surface plane 118 of the substrate(e.g., fully retained cells and particles), and egress cells and/orparticles that traverse the surface plane 118 of the substrate (e.g.,partially retained cells and particles). In such variations, any cellsand/or particles that are not fully retained by their associated wellscan be egressed in subsequent re-distribution steps as described insections below. In a preferred variation, the well dimensions areselected to retain a single cell and a single particle as acell-particle pair within the well cavity 128 and below the surfaceplane 118, however, the well dimensions can be configured to retain anynumber or combination of cells and non-cell particles, and can beutilized by method 200 in any other suitable way.

2.1 Method—Receiving the Population of Target Cells

Block S210 recites receiving a population of target cells into an arrayof wells. Block S210 functions to receive a biological sample includingtarget cells of interest at an embodiment of the system 100 described inSection 1 above, and to facilitate distribution of the target cells intowells of the system 100 in at least one of single-cell format andsingle-cluster format. However, Block S210 can alternatively includereceiving a biological sample at any other suitable system configured tocapture cells in at least one of single-cell format and single-clusterformat. In variations of Block S210, the biological sample can bereceived directly at a variation of the array (e.g., by pipetting, byfluid delivery through a fluid chanel coupled to the array, etc.), byway of a variation of the first plate of a fluid delivery module (e.g.,through a fluid reservoir 160 defined by a recess of the first plate,from a fluid channel embedded within the first plate and in fluidcommunication with the array, etc.), and/or in any other suitablemanner. Furthermore, in variations of Block S210, the cell populationcan include a cell population of target cells (e.g., CTCs, CSCs, Immunecells) and/or any other suitable particle of interest.

In variations of Block S210, as shown in FIG. 21, receiving thepopulation of target cells can include distributing the population oftarget cells to the array of wells, and can optionally includere-distributing the population of target cells in one or more additionalsteps to correct for inaccuracies in the initial distribution step(e.g., when a subset of wells receives more or less cells than desired,when a subset of cells aggregates at a region of the substrate, etc.),which can improve the efficiency of capturing a desired quantity oftarget cells in each well. In a preferred embodiment, coordination ofBlock S210 with the size constraints of the well cavities of the arrayof wells can determine the number and shape of cells that can becaptured and retained within the wells, as further described in BlockS218 below. For example, a well with a height of 30 micrometers canreceive one target cell with a characteristic diameter between 15-25micrometers below the surface plane 118, and any additional target cellsthat enter the well beyond the first target cell traverse the surfaceplane 118, and exceed the boundaries defined by the height of the wellcavity 128. Accordingly, any cells that are not fully retained by thewell cavity 128 but instead traverse the surface plane 118 can beegressed from the well cavity 128 by a subsequent cell re-distributionstep, by which a subset of wells of the array of wells that receive morethan one cell (e.g., in a cell-saturated state) can be corrected tocontain only a single cell by the redistribution step. However,receiving the population of target cells can include any method orsequence of distribution and re-distribution steps to achieve thedesired contents within each well.

Block S210 is preferably performed as a first step of method 200, priorto any other cell or particle distribution steps, and wherein the arrayof wells has no other cell or non-cell particle inside the wells (e.g.the wells are in an unoccupied state prior to Block S210). However,Block S210 can be performed at any other suitable time, such asfollowing distribution of the population of particles in Block S220,and/or repeated after a first cell distribution step. Furthermore, BlockS210 or substeps within Block S210 can be repeated any number ofsuitable times in order to achieve desired distribution of target cellsinto the array of wells.

Distributing the population of target cells into the array of wells alsofunctions to maximize cell viability during the capture of the targetcells into the wells. In a first variation, an initial distribution stepfor the population of target cells can include loading the cells intothe wells by gravity-induced entry, without additional physical forcesapplied to the cells. In an example, gravity-induced entry of cells intothe array of wells can be achieved by pipetting small aliquots (e.g.,ranging from 50-500 ul per aliquot) of the sample at different regionsof the array, and incubating the biological sample at the open surfaceof the array (at which the open ends of each well are located) for analotted period of time to allow target cells to enter the wells bygravity. More specifically, each aliquot can be delivered to a differentregion of the array of wells within a fluid reservoir superior to thearray of wells to distribute the sample across the open surfaces of eachwell, generating a fluid layer sitting above the array of wells fromwhich cells can descend into the wells. In variations, time periodsallotted for gravity-induced entry of single target cells into the arrayof wells can range from 5 minutes to 60 minutes for a volume of 1 mL(e.g., approximately 20 minutes for K562 cells, and approximately 30minutes for PBMCs). However, gravity-induced entry of cells can also beachieved by any other suitable method, such as: smearing the biologicalsample at the array of the substrate, maintaining a stable fluid layerof the biological sample within the fluid reservoir at a consistent,steady-state velocity, and/or in any other suitable manner of sampledeposition and distribution that minimizes application of additionalphysical forces onto the target cells.

In a second variation, distribution of the population of target cellscan include application of force to accelerate the speed of cell capturebeyond the forces of gravity alone. In one example, distribution of thetarget cells can be achieved by cytospinning the substrate with thebiological sample about an axis parallel to the broad surface of thesubstrate, cytospinning the substrate with the biological sample aboutan axis perpendicular to the broad surface of the substrate, orcytospinning the substrate with the biological sample about an axisoriented at any suitable angle relative to the broad surface of thesubstrate. Furthermore, in applications of Block S210 includingcytospinning, an axis of rotation can be offset from any suitablereference point of the substrate, in any suitable manner. In anothervariation, distribution of the sample across the open surfaces of thearray of wells can be achieved by gently tipping or rocking thesubstrate about a transverse axis passing through the midpoint of thesubstrate, or any other suitable axis of rotation. However, capturingthe cells can include any other suitable method for sample distributionto encourage efficiency and speed of cell entry into the wells, whileminimizing cell damage.

Block S210 can optionally include an additional re-distribution step toensure optimal distribution of cells and maximize the number of targetcells that are captured in single-cell format. In a preferredembodiment, as shown in FIG. 21, re-distribution comprises flowing acell distribution fluid along a fluid path through a fluid reservoirparallel to the surface plane 118, wherein the fluid reservoir spans thearray of wells along the open ends of the wells at the surface plane118. In one variation, re-distributing can impart a force from the celldistribution fluid to a subset of partially retained cells (crossingover the surface plane 118), egressing the partially retained cells outof their respective cell-saturated wells, and transmitting the paritallyretained cells downstream of the array of wells to wells that arecapable of receiving cells (e.g., in an unoccupied state,cell-accessible state). However, re-distributing can additionally and/oralternatively function to transmit any subset of cells across the arrayof wells, with any position relative to the array of wells (e.g., cellsthat are above the surface plane 118 and distal from the array of wellswithin the fluid reservoir), to wells in any other occupied state (e.g.,wells containing one or more cells, wells containing one or morenon-cell particles). In a preferred application, re-distribution inBlock S210 can improve single-cell capture efficiency, wherein at least50% of the cells within the population of target cells are captured insingle-cell format.

The cell distribution fluid used for cell re-distribution can be anysuitable fluid. In one variation, the cell distribution fluid has adensity less than the solution within the well cavities of the array ofwells, such that when the cell distribution fluid is flowed across thefluid path superior the array of wells, the cell distribution fluid doesnot readily enter the well cavity 128 of the wells, and therefore canonly egress cells that cross the surface plane 118 of the substrate, andprotrude into the fluid path. However, the cell distribution fluid canhave any suitable density or characteristic that can assist intransmitting partially retained cells or cells above the surfaceboundary of the substrate downstream of the fluid path.

Furthermore, the flow rate and flow direction of the cell distributionfluid can be modulated and controlled. In a preferred variation, theflow rate and flow direction can be controlled by a fluid deliverymodule comprising a flow control subsystem that can apply a net positiveor a net negative pressure at either side of the fluid reservoir throughwhich the cell distribution fluid can flow. The flow direction can beunidirectional along the fluid path, but can additionally and/oralternatively be bidirectional, multidirectional, randomized, and/or atany angle relative to the fluid path to permit exposure of the cellswithin the cell distribution fluid to the open ends of cell-accessiblewells. In one example, re-distribution can include at least one flowcycle of the cell distribution fluid wherein the flow directionalternates between a first forward direction and a second reversedirection opposing the forward direction, wherein partially retainedcells that are egressed from the wells by fluid flow in the firstforward direction can be washed back towards the array of wells by fluidflow in the second reverse direction in order to access cell-accessiblewells of the array of wells. The number of redistribution flow cyclescan be any suitable number of cycles necessary such that at least amajority of the target cells in the population of cells is retainedbelow the surface plane 118 of the substrate within the array of wells.In a preferred application, re-distribution results in a maximum numberof target cells of the population of target cells being captured insingle-cell format within the array of wells. However, the celldistribution fluid flow rate and flow direction can be otherwiseconfigured.

In a preferred application, as shown in FIG. 21, the wells of the arrayof wells are configured to permit a maximum of a single cell to be fullyretained by the well below the surface plane 118. As such, anyadditional cell entering the well beyond the first fully retained cellwill not be permitted to descend below the surface plane 118. Instead,the additional cell will protrude from the open end of the well,traversing the surface plane 118 between the well cavity 128 and thefluid path and thereby becoming accessible by the cell distributionfluid during a subsequent re-distribution step. In a first variationwherein Block S210 is performed as the first step of method 200 andwherein each well of the array of wells is in an unoccupied statecontaining no other cell or non-cell particle, the initial distributionstep(s) for receiving the population of cells into the array of wellscan result in any output combination of a first subset of wells of thearray of wells receiving a single cell, defined herein as aparticle-accessible state or a single-cell state; a second subset ofwells receiving more than one target cell, defined herein as acell-saturated state; and a third subset of wells receiving no targetcells, defined herein as an unoccupied state (FIG. 21). Upon completionof the initial distribution step, only the first subset of wells hassuccessfully captured single cells in each well and are useful forsubsequent processing steps of the preferred application involvingsingle-cell capture. In order to increase the number of wells in thefirst subset of wells (to increase the efficiency of single cell captureinto the array of wells), additional cells localized in the secondsubset of particle-saturated wells can be optionally re-distributed towells in the third subset of unoccupied wells, using an additionalre-distribution step. In a second variation, the initial distributionstep(s) for receiving the population of cells can result in an outputcondition wherein a subpopulation of cells remains above the surfaceplane 118 of the substrate and within the fluid reservoir. In order toutilize the subpopulation of cells that are available to populate thearray of wells and increase the number of wells in the first subset ofwells, the sub-population of cells can be optionally re-distributed towells in third subset of unoccupied wells, using the additionalre-distribution step.

Block S210 can be performed at a low temperature, using the thermalcontrol module described in Section 1. In a preferred variation, thetemperature of the array of wells is maintained at less than 10° C.,however Block S210 can additionally and/or alternatively be performed atany other suitable temperature to maintain the viability of thepopulation of target cells within the array of wells.

In order to facilitate efficient capture of the population of targetcells in single cell format, Block S210 can be performed using abiological sample containing a specific ratio of target cells to thenumber of wells in the array of wells. In one variation, the number oftarget cells in the biological sample is preferably less than 20% of thenumber of wells in the array of wells. Preferably, the ratio of thenumber of cells in the sample to the number of wells in the array can beas low as 1:10, though the ratio can be any other suitable ratio. In aspecific example, for an array containing 200,000 wells, Block S210 canbe performed using less than 20,000 target cells in the biologicalsample solution, (e.g., approximately 2,500 cells, 5,000 cells 10,000cells, 15,000 cells, etc.). However, Block S210 can be performed usingany suitable number of cells in the sample in relation to the number ofwells in the array of wells, such as 150,000 cells in 200,000 wells, or250,000 cells in 200,000 wells. Furthermore, capturing the cells caninclude any other suitable concentration of cells in the sample in orderto encourage single cell capture.

Block S210 can additionally include preparing the biological samplecontaining the population of target cells S212 prior to the distributionof the biological sample to the array of wells. In variations, BlockS212 functions to enhance the efficiency of target cell capture withinthe array of wells by increasing the concentration of target cellswithin the sample, and can comprise one or more of: spiking cells intothe biological sample, combining a pre-fixing solution with thebiological sample, adding saline to the biological sample, anddelivering the biological sample into the array of wells; however, BlockS212 can additionally or alternatively comprise any other suitablebiological sample preparation step. For instance, the biological samplecan include a cell population of interest as the target cell population,thus eliminating a need for spiking cells into the biological sample.The biological sample can include a raw biological sample (e.g., blood,urine, tissue), enriched cell lines (e.g., isolated from blood), oraugmented target cells (e.g., bound to small particles, pre-labeled witha marker). In a first variation, the biological sample is an enrichedpopulation of cancer stem cells isolated from blood. In a secondvariation, target cells (e.g., T-cells, B-cells, cancer stem cells) arebound to a small particle to selectively increase effective cell size.In a third variation, target cells are prelabeled with a fluorescentlabel, gold nanoparticle, or chemical linker to aid in downstreamidentification and/or quantification of the cells. However, thebiological sample can include any other suitable component and bepre-processed in any suitable manner.

In an example, using a specific example of the system 100 describedabove, Block S212 can comprise delivering the biological sample (e.g., 2mL of whole blood), with a cell population of interest (e.g., MCF7breast cancer cells, SKBR3 breast cancer cells, LnCAP prostate cancercells, PC3 prostate cancer cells, HT29 colorectal cancer cells) preparedwith or without cell spiking, to the fluid reservoir 160 and rotatingthe cylindrical cartridge of the fluid delivery module, such that achamber containing an appropriate biological sample preparation solutioncan be punctured by the actuation system. The biological samplepreparation solution can then flow into the fluid reservoir 160, to becombined with the biological sample, and then be delivered into thearray of wells upon pressure generation by the pump of the flow controlsubsystem. Block S212 can, however, comprise any other suitable methodof preparing a biological sample, including the target cell population,to be received by the array of wells at the substrate.

Block S210 can additionally include priming the substrate in Block S214prior distribution of the biological sample to the array of wells, whichfunctions to minimize trapped air and/or cell aggregates within thefluid channels of the substrate, and to prepare the system for receivinga biological sample including the target cell population. Priming thesubstrate includes flowing a priming reagent or buffer solution throughthe fluid channels of the substrate. In an example, delivering a buffersolution into the array of wells comprises delivering a buffercomprising 1% bovine serum albumin (BSA) and 2 mMethylenediaminetetraacetic acid (EDTA) in ix phosphate buffered saline(PBS); however, delivering a buffer solution can comprise delivering anyother suitable fluid into the array of wells. In the example, using aspecific example of the system 100 described above, priming thesubstrate in Block S214 can comprise rotating the cylindrical cartridgeof the fluid delivery module, such that a chamber containing the buffersolution can be punctured by the actuation system. The buffer solutioncan then flow into the fluid reservoir 160, to be delivered into themanifold and into the microfluidic chip upon pressure generation by thepump. The buffer solution can then be driven in a forward direction anda reverse direction, by the pump, to adequately remove bubbles from thearray of wells. Block S214 can, however, comprise any other suitablemethod of delivering a buffer solution into a array of wells configuredto capture the target cell population.

In another variation, priming the substrate can function to sterilizethe substrate. In an example, sterilization of the substrate can includeadding a total of 800 ul of 100% ethanol to the substrate, followed byincubating the substrate under ultraviolet light (e.g., via the imagingsubsystem 194 as described in Section 1) for approximately 5 to 20minutes to sterilize the substrate. However, the substrate can be primedprior to the distribution of cells and/or non-cell particles in anyother suitable manner.

After capturing the population of target cells, Block S210 canoptionally include gathering information from the captured cells,including identifying, quantifying, and locating the captured cells inBlock S216. Block S216 is preferably achieved using the imagingsubsystem 194 described in Section 1, but can be achieved using anyother method and/or component of the system 100. The informationobtained in Block S216 can further be used to inform, modify, and/oradjust settings for subsequent or concurrent steps in method 200. In onevariation, the captured population of target cells can be phenotypicallycharacterized and/or quantified by staining the captured cells withfluorescent antibodies (e.g., directly or indirectly labeled). Forexample, specific stains can be used to quantify the number of viablecells, specific states of the cells in their cell cycles and/orsub-types of the target cells. Furthermore, imaging of the array ofwells via the imaging subsystem 194 can also be used to ascertain theexact number of captured cell-particle pairs so that the appropriateparmeters for the downstream library preparation and/or next-gensequencing can be determined (e.g., to determine a number of PCRamplification cycles and/or sequencing depth). In a first example,information regarding the number of cells that have been successfullycaptured in single-cell format can be used to determine and select thenumber of particles (e.g., increasing or decreasing concentration ofparticles in solution) required for efficient distribution and singlecell-particle capture in Block S220. In a second example, informationindicating that a majority of cells in the sample have already beencaptured successfully in single-cell format can be used to instruct thesystem to skip and/or adjust an additional re-distribution step and/orprocessing step, thereby decreasing processing time and enhancingperformance. Alternatively, information indicating cell aggregation orinefficient cell capture (e.g., a majority of uncaptured cells remainingin the fluid reservoir) can instruct the system to include an additionalre-distribution step and/or processing step. In a second variation,information regarding individual locations of captured cells and theirrelative distribution within the array of wells can be used to instructcomponents of the fluid delivery module described in Section 1 to adjustsettings for more efficient capture. In one example, informationindicating that a majority of captured cells are located at a first sideof the substrate in comparison to a second side of the substrate caninform the flow control subsystem to select a specific flow rate andflow direction to flow uncaptured and/or partially retained cellstowards the second side of the substrate during a re-distribution step.In another example, location information of wells containing singlecells (e.g., particle-accessible wells) can be used by the flow controlsubsystem to select a specific protocol for distributing the populationof particles in Block S220 in order to enhance the probability thatparticle-accessible wells receive particles to achieve an ideal state.However, information regarding the captured cells is not limited toquantification and location within the array of wells, and can be usedto instruct and modify any of the steps in method 200. Furthermore,Block S216 can be repeated any number of times throughout method 200,and can be performed before, after, and/or during any step of method200, including to assess and instruct the performance of individualsteps of cell distribution in Block S210, particle distribution in BlockS220, and particle re-distribution in Block S230.

Block S210 can optionally include selecting an array of wells comprisingwells with a specific dimension, geometry, density, and/or spatialarrangement within the substrate in Block S218, according to at leastone of: the dimensions and numerosity of the desired target cells, thedimensions and numerosity of the particles used in Block S220, thecapture assay or protocol performed, and the desired output conditionfor the well state upon completion of at least a portion of method 200,including the number of cells and/or particles desired to be captured ineach well. The wells of the array of wells can posses a range ofdimensions that can impact the output states of wells upon completion ofBlock S210 and/or Block S220, including horizontal cross-section,vertical cross-section, width of the open end of each well, height ofthe well cavity 128, and total volume of the well cavity 128. In onevariation, for which it is desired that an ideal state of the wellcomprises receiving exactly one cell and one particle into the samewell, the dimensions of the well can be sufficient enough to retain boththe cell and the particle below the surface plane 118 (e.g., the openend of the well), but not sufficient enough to retain a second cellafter a first cell has been received in Block S210 (resulting incell-saturated state), or more than one particle after a first cell hasbeen received in Block S210 (resulting in a particle-saturated state).In a specific example, for a population of target cells comprisingtarget cells having a characteristic diameter between 10-15 micrometersand a population of particles having a characteristic diameter between18-22 micrometers, the height of the well cavity 128 can range between20 and 50 micrometers, and the width of each well (e.g., horizontalcross section) can range between 20 and 30 micrometers. In anotherexample, for which it is desired that an ideal state of the wellcomprises receiving either exactly one cell (e.g., between 10-15micrometers) or one particle (e.g., between 18-22 micrometers) (but notboth a cell and a particle), the height of each well cavity 128 canrange between 10 and 30 micrometers, and the width of each well (e.g.,horizontal cross section) can range between 20 and 30 micrometers.However, the dimensions of the wells can be selected based on any othersuitable criteria and can be matched and correlated to the dimensions ofthe population of cells and/or dimensions of the population of particlesused in any step of method 200.

2.2 Method—Distributing the Population of Particles

Block S220 recites: distributing a population of particles into thearray of wells. Block S220 preferably functions to colocalize a singleparticle of the population of particles with a single target cellpreviously retained within a particle-accessible well (e.g., in thefirst subset of wells) described in a variation of Block S210, therebycapturing a single cell-particle pair within individual wells. However,Block S220 can be performed before or after any other step in method200, and can be used to distribute any number of particles intoindividual wells of the array of wells, including wells previouslycontaining any number of cells and/or non-cell particles, and/or intowells that are previously unoccupied. In a preferred application, as aresult of consecutive steps in Block S210 and Block S220, the targetcell and the particle are fully retained by the well cavity 128 of thewell (e.g., the cell-particle pair is retained below a surface plane 118of the substrate), such that the particle comprising the cell-particlepair is not egressed from the well cavity 128 during re-distribution ofparticles in Block S230. However, Block S220 can be implemented in anyother suitable manner in method 200, in order to distribute thepopulation of particles across the array of wells, and to control thenumber of particles received and retained into individual wells of thearray of wells.

Preferably, Block S220 is performed after receiving the population oftarget cells at the array of wells as described in Block S210, such thatparticles added to a well that is currently occupied by a target cellsettle on top of the target cell and proximal the open surface of thesubstrate, however Block S220 can be additionally and/or alternativelyperformed prior capturing target cells at the array of wells, and intemporal relation to any other suitable step of method 200. To ensurethat the population of particles is distributed uniformly into the arrayof wells and encourage a 1:1 ratio of target cell to particle per well,Block S220 can be followed by Block S230, wherein particles areredistributed across the array of wells, and analogous to redistributionof cells described in Block S210. However, Block S220 can include anyother suitable steps.

In Block S220, the population of particles (e.g., microspheres, beads,etc.) received into the array of wells can be of any suitable materialto convey desirable properties to the particles, including physicalproperties (e,g., nonswelling behavior, dissolveability), magneticproperties, optical properties, chemical properties (biocompatibility,binding affinities), and thermal properties. The particles can be madeof polystyrene, silica, non-porous glass, porous glass, coated glass,and/or a combination of one or more suitable materials. The density ofthe particles is preferably greater than the buffer of the containingsolution (e.g., at least 1.1 g/cc) but can alternatively be of any othersuitable density.

Preferably, the particle has a characteristic dimension configured suchthat only a single particle can enter a well currently occupied by asingle target cell below the surface plane 118, in order to colocalizethe single cell-particle pair within an individual well. In a preferredvariation, each particle in the population of particles is a sphericalparticle including an outer shell with a diameter between 10 to 30micrometers. To accommodate facile distribution and settling into thearray of wells, the population of particles can be produced with asubstantially monodisperse geometry. In examples, the population ofparticles can posses a characteristic dimension including a diameter ofone of: 20 microns, 15 microns, 30 microns, 35 microns, and/or 40microns, with a standard deviation of less than 20% or less than 15%,thus improving the efficiency of single cell-particle pair capture toabove 50%. In addition, the uniformity of the population of particlescan enable a higher percentage of particle retrieval upon completion ofvarious steps of the single cell sequencing preparation biochemicalprocesses (in Block S250). To minimize sedimentation and improvedistribution, the solution containing the population of particles canoptionally include approximately 10% glycerol.

In a specific example of this variation, the particles are glass beadswith a polystyrene coating approximately 20 micrometers in diameter(e.g., 15 to 25 micrometers), but can alternatively and/or additionallybe any other suitable diameter. Furthermore, the particles can be anyother suitable 3-dimensional (e.g., rectangular, triangular, oblong, rodor any other polygonal shape) or 2-dimensional shape (e.g., a sheet). Ina specific example, wherein the height of the well is approximately 40micrometers, and wherein the target cell has a characteristic dimensionthat ranges between 15-25 micrometers, particles with a diameter between18-22 micron can be used in Step S220 to colocalize a single particlewith a single captured cell below the surface plane 118. However,implementation of method 200 by system 100 can be configured in anyother suitable manner.

In variations of Block S220, the outer shell of the population ofparticles can include various surface properties and/or surface featuresthat function to interact with the captured target cells, biomoleculesof the captured target cells, the interior surface 130 of the wellcavity 128, the solution within the well, and/or any other suitablecomponent of the system and/or method described in this application.

In a first variation, as shown in FIG. 20, the outer shell of theparticles can be conjugated to a probe and/or set of probes 36 (e.g.,oligonucleotide, chemical linker, etc.) that preferably function to bindto intracellular nucleic acid content (e.g., mRNA, DNA, proteins, etc.)released from the cell and additionally or alternatively to facilitatedownstream processes (e.g., reverse transcriptase, polymerase chainreaction (PCR), etc., as described in variations of Block S240. However,the set of probes 36 can be implemented in any other manner in order toperform analysis of the target cells. In variations wherein the probecomprises a biomolecular interaction region comprising a nucleotidesequence, the biomolecular interaction region of individual probes canadditionally or alternatively contain any combination of at least oneof: a primer sequence (e.g., a PCR handle), a cell barcode used toidentify the cell from which the nucleic acid content originated (e.g.,a unit identifier), a unique molecular identifier (UMI) to labeldifferent molecules (e.g., genetic material) of the same cell (e.g., onebarcode can have multiple UMIs), a poly-DT sequence which enablescapture of polyadenylated mRNA species, and surface hydroxyls reactedwith a polyethylene-glycol (PEG) derivative to serve as a support foroligo synthesis. In addition to the biomolecular interaction region,each probe can include a particle linking region including a particlelinker that couples the probe to the particle, and additionally oralternatively a substrate linking region including a functional linkerthat binds to a portion of a cell or portion of a surface of a well(e.g., via protein, antibody affinity, chemical interactions) (FIG. 20,FIG. 7). In a specific example, a single particle coupled to a singleset of probes 36 on the outer shell of the particle is received into thewell cavity 128 of an individual well during Block S220. The set ofprobes 36 is coupled to the outer shell of a particle by a particlelinker containing a photo-cleavable bond that can be activated usingultraviolet (UV) wavelengths. Under UV irradiation, the set of probes 36can be controllably detached from the particle within the well, and theparticle can be optionally removed from the well cavity 128, therebyallowing the set of probes 36 to interact with the environment withinthe well cavity 128 without the physical constraints of the particle.However, the biomolecular interaction region, the particle linkingregion, and substrate linking regions of the set of probes 36 can beotherwise configured.

In another variation, the outer shell of each particle can be modifiedto tailor the linker density of barcoded oligonucleotides to a specificnumber in order to maximize the number of biomolecules captured from thesingle cell lysate, while minimizing effects of steric hindrance duringdownstream processing of genetic complexes formed with the probe (e.g.,during Block S240), such as cDNA reverse transcription, and/or PCRamplification. For single cell RNA-seq, the particles can be tailored tohave a linker density between 0.1 to 5 micro-mole/gram. For example, thelinker density on the particles can be tailored such that the maximumnumber of biomolecules from a single cell lysate can be one ofapproximately: 100,000 molecules, 500,000 molecules, 750,000 molecules,1,000,000 molecules, 2,000,000 molecules, 3,000,000 molecules, 4,000,000molecules, and/or 5,000,000 molecules. Prior to oligonucleotide binding,the outer surface area and/or porosity of the particles can be tailoredby various processes such as chemical etching, chemically growing a thin(up to few, 0.1, 1, 2, and/or 3 micron in thickness) layer offunctionalized surface. In another variation, the outer shell of theparticles can include multiple and distinct functional groups ofpredetermined density to be able to bind different types of biomoleculesfrom the single cell or single cell lystate, such as: DNA, mRNA,proteins, metabolites, glycans, cellular enzymes, etc. However, thefeatures and characteristics of the outer shell of the population ofparticles can be configured in any other suitable manner.

Furthermore, the surface features of the population of particles can beotherwise configured to perform any other suitable function orinteraction within the array of wells. In a second variation, the outershell of the particles can include surface-bound moieties able to bindto an antibody or any other suitable protein either on the cell or onthe wall of the well. In a third variation, the outer shell of theparticles can contain synthetic or organic materials that improve thebiocompatibility of the particle (e.g., PEG, collagen, etc.). In afourth variation, the outer shell of the particles can contain aphysical feature that allows the particle to enter and be retainedwithin a well, such as a hook, adhesive, or extendable volume. However,the outer shell of the particles can be otherwise configured.

Furthermore, each particle of the population of particles can optionallycontain, within the particle, a synthetic reagent, biochemical agent,and/or an organic material that can be used to conduct biochemicalassays on the captured cell. In a variation, the particles can bemanufactured to contain a drug that can be released from the particle(e.g., triggered by time, temperature, pH, etc.), with applications forhigh-throughput drug testing on single cells within the array of wells.For example, the outer shell of the particle can be composed of abiodegradable material the dissolves over time at a certain temperature,thereby releasing the drug in the presence of the target cell within thewell. However, the population of particles can be configured in anyother suitable manner.

To perform Block S220, the ratio of the number of particles to thenumber of wells is at least 1:1 and is preferably at least 1.5:1, butcan be any other suitable number of particles. In a variation whereincapture of a single cell-particle pair is desired, the number ofparticles within the particle solution is selected such that every wellthat contains a single cell receives at least one particle. In aspecific example, wherein the number of wells in the array is 200,000,the number of particles added to the array preferably ranges between300,000 to 400,000 particles. Furthermore, as previously described,obtaining information about the number of cells captured in single-cellformat upon completion of Block S210 can be used to assist in selectionof a range of number particles within the particle solution, to enhancethe desired capture efficiency, and minimize under or over-saturation ofthe array of wells with particles. In one example, upon determining thata range of 5000 to 6000 wells of an array of 200,000 wells are in aparticle-accessible state, the number of particles in the particlesolution can be selected within a range of 10,000 to 50,000 particles tominimize occurrences of particle-saturation states, thereby improvingcolocalization efficiency and/or speed of a single particle to a singlecell. However, selecting the number of particles added to the array ofwells in Block S220 can be performed in any other suitable manner.

Distributing the population of particles into the array of wellsincludes an initial distribution step which functions to load theparticles into the array of wells, similar to initial distribution ofthe cells, as described in Block S210. In variations, the population ofparticles can enter the array of wells by gravity-induced entry (e.g.,pipetted into the array in multiple aliquots), with an applied force,(e.g., cytospinning the substrate with solution containing thepopulation of particles), and/or flowed into the fluid reservoir 160 bythe reagent delivery module as described in Section 1. In a specificexample, a particle solution containing a range of between 200,000 to600,000 particles can be distributed to the array of wells by pipettingsmall aliquots of the solution at various regions of the array of wellsto form a uniform fluid layer within the fluid reservoir superior theopen ends of the wells, followed by centrifuging the array of wells at330 rpm for 4 minutes. However, initial distribution of the particlescan be performed by any other suitable manner using any appropriatecomponent of the system 100.

In a preferred application wherein Block S220 is performed after BlockS210, the initial distribution step(s) for receiving the population ofparticles into the array of wells can result in any output combinationas shown in FIG. 22, including: a first subset of wells of the array ofwells retaining a single cell receiving a single particle, definedherein as an ideal state containing a single cell-particle pair; asecond subset of the array of wells retaining a single cell receivingmore than one particle, defined herein as a particle-saturated state; athird subset of unoccupied wells receiving only a single particle,defined herein as a cell-accessible state, and a fourth subset ofunoccupied wells receiving more than one particle (FIG. 22). Uponcompletion of the initial distribution step, only the first subset ofwells in the ideal state has successfully captured a singlecell-particle pair in each well and are useful for subsequent processingsteps of the preferred application involving single-cell and/or singlecell-particle pair capture. In order to increase the number of wells inthe first subset of wells in the ideal state (to increase the efficiencyof single cell-particle capture into the array of wells), additionalparticles localized in the second subset of particle-saturated wells canbe optionally re-distributed to wells that remain unoccupied after theinitial distribution step and/or wells that remain in theparticle-accessible state (e.g., containing only a single cell) afterthe initial distribution step, as described in Block S230. Additionallyand/or alternatively, partially retained particles and/or excessparticles remaining in the fluid reservoir can be egressed from thefluid reservoir through an outlet coupled to a waste chamber. However,Block S220 can be performed in any other suitable manner.

2.3 Method—Redistributing the Population of Particles

In Block S230, redistributing the population of particles functions toensure optimal distribution of particles across the array of wells, andto maximize the number of particles that can access the array of wells,preferably to enable single cell capture and/or single cell-particlepairing within individual wells. Block S230 can redistribute particlesthat have been added to the array of wells, but have not been fullyretained within individual wells (e.g., into the well cavity 128, belowthe surface plane 118), including particles that remain in the fluidreservoir above the surface plane 118 (e.g., excess particles), andparticles that have entered individual wells, but exceed the volumecapacity of the well and traverse the surface plane 118 into the fluidpath. In a preferred embodiment, similar to the cell re-distributionstep of Block Sllo, particle re-distribution comprises flowing aparticle distribution fluid along a fluid path through a fluid reservoirparallel to the surface plane 118, wherein the fluid reservoir spans thearray of wells along the open ends of the wells at the surface plane118. In one variation, as shown in FIG. 22, re-distributing can impart aforce from the particle distribution fluid to a subset of partiallyretained particles (crossing over the surface plane 118), egressing thepartially retained particles out of their respective particle-saturatedwells, and transmitting the partially retained particles downsteam ofthe array of wells to wells that are capable of receiving particles(e.g., in an unoccupied state, particle-accesible state). However,re-distributing can additionally and/or alternatively function totransmit any subset of particles across the array of wells, with anyposition relative to the array of wells (e.g., particles that are abovethe surface plane 118, particles that are distal from the array of wellswithin the fluid reservoir), to wells in any other occupied state (e.g.,wells containing one or more cells, wells containing one or morenon-cell particles).

The particle distribution fluid used for particle and/or cellre-distribution can be any suitable fluid, or a fluid containing anysuitable component to flow across the upper surface of the array ofwells. In one variation, the particle distribution fluid has a densityless than the solution within the well cavities of the array of wells,such that when the particle distribution fluid is flowed across thefluid path superior the array of wells, the particle distribution fluiddoes not readily enter the well cavity 128 of the wells, and thereforecan only egress particles that cross the surface plane 118 of thesubstrate, and protrude into the fluid path. However, the distributionfluid can posses any other suitable characteristic. In anothervariation, wherein the fluid reservoir includes a set of magneticelements (e.g., along the fluid path), the particle distribution fluidcan contain a set of magnetic particles that can be used to controland/or concentrate the flow of the distribution fluid across a specificspatial region relative to the array of wells (e.g., only through thefluid path proximal the magnetic elements in the fluid reservoir).However, the distribution fluid can posses any other functionalcomponents that can modify the flow rate and/or flow direction of thedistribution fluid (e.g., electrostatic, physical, chemical, thermalproperties). Furthermore, the particle distribution fluid can have anysuitable density, characteristic, and/or contain components that canassist in transmitting partially retained particles or excess particlesabove the surface plane 118 of the substrate downstream of the fluidpath.

Preferably, the flow rate and flow direction of the particledistribution fluid can be modulated and controlled. Similarly to apreferred variation in Block Sllo, the flow rate and flow direction canbe controlled by a fluid delivery module comprising a flow controlsubsystem that can apply a net positive or a net negative pressure ateither side of the fluid reservoir through which the particledistribution fluid can flow. The flow direction can be unidirectionalalong the fluid path, but can additionally and/or alternatively bebidirectional, multidirectional, randomized, and/or at any anglerelative to the fluid path to permit exposure of the particles withinthe particle distribution fluid to the open ends of particle-accessiblewells. In one example, re-distribution can include at least one flowcycle of the particle distribution fluid wherein the flow directionalternates between a first forward direction and a second reversedirection opposing the forward direction, wherein partially retainedparticles that are egressed from particle-saturated wells by fluid flowin the first forward direction can be washed back towards the array ofwells by fluid flow in the second reverse direction in order to accessparticle-accessible and/or unoccupied wells of the array of wells. Thenumber of redistribution flow cycles can be any suitable number ofcycles necessary such that at least a majority of the particles in thepopulation of particles is retained below the surface plane 118 of thesubstrate within the array of wells. In a preferred application,re-distribution can result in at least 80% of particle-accessible wellsbeing filled with a single particle to achieve an ideal state for thewells. However, the cell distribution fluid flow rate and flow directioncan be otherwise configured.

As described in Block S216, the fluid flow rate and flow direction ofcell and/or particle distribution fluids in Block S210, Block S220, andBlock S230 can be modified and/or adjusted accordingly, and/or in realtime during synchronous or asynchronous steps of method 200, accordingto the quantity and location of captured cells within the array ofwells. In a preferred variation, the optical subsystem can record a setof images to obtain information regarding the abundance and distributionof particle-accessible wells, and can communicate instructions to thefluid delivery module and/or flow control subsystem to assist in theselection of at least one of: concentration of particles in the particlesolution, flow rate of the particle distribution fluid, direction of theparticle distribution fluid, temperature of the particle distributionfluid, and/or number of repeated cycles of re-distribution, in order toenhance capture efficiency. However, the parameters of redistribution inBlock S230 can be manually determined by user input (before, during,and/or after one of Block S210, Block S220, Block S230), statically set(pre-determined, according to a stored setting), and/or otherwisedetermined. In another preferred embodiment, the extraction module canbe used in operation with the imaging subsystem to retrieve one or moreparticles from a well containing more than one particle and dispensethem into another well containing no beads, based on imaging feedback.

2.4 Method—Processing the Array of Wells

Block S240 recites: processing the array of wells, which can include oneor more of: receiving at least a single process reagent at the array,thereby facilitating diffusive delivery of one or more process reagentsto the cell population in at least one of single-cell format andsingle-cluster format, performing a biochemical process at the array ofwells, analyzing the contents of the array of wells, and additionallyand/or alternatively any other suitable process at the array of wells.Block S240 functions to enable seamless and rapid processing andanalysis of the contents captured within the array of wells, withoutnecessitating removal of the captured contents (e.g., cells and/ornon-cell particles) from the array of wells, and furthermore can be usedto process multiple arrays of wells simultaneously (e.g., 2, 4, 6, 10arrays at a time). In preferred embodiments, portions of Block S240 canbe performed automatically in coordination with components of system100, wherein parameters of process reagent delivery including thesequence of multiple reagents, timing (e.g., dispense time, flowduration), velocity, direction, and/or volume of process reagents (e.g.,using the fluid delivery module), parameters for temperature modulationof the array of wells and/or process reagents (e.g., using the thermalcontrol module), and/or parameters for optical analysis (e.g., using theoptical subsystem) can be determined, selected, and/or coordinated usingstored settings, user input, and/or adaptive input and one or moreprocessors of system 100. However, processing the array of wells caninclude any other suitable steps and/or parameters, and can be performedin any other suitable manner.

In variations, delivering one or more process reagents to the array ofwells can function to perform one or more of: permeabilizing capturedcells of the target cell population, post-fixing captured cells of thetarget cell population, blocking captured cells of the target cellpopulation, washing captured cells of the target cell population,treating captured cells of the target cell population with an antibodycocktail, incubating captured cells of the target cell population,staining captured cells of the target cell population, lysing capturedcells of the target cell population, isolating components withincaptured cells of the target cell population, and heating the array ofwells. Step S240 can additionally or alternatively comprise any suitablestep that prepares the cells of interest captured by the array of wellsfor analysis, such as delivering a hybridization buffer to the cells ofinterest, delivering particles and/or control probes to the cells ofinterest, dehydrating the cells of interest, and/or denaturing the cellsof interest. In one variation, Step S240 can prepare cells of the targetcell population for an analysis requiring a stain (e.g. fluorescentstain or histological stain). In another variation, Step S240 canprepare cells of the target cell population for an analysis involvingelectrophoresis. In yet another variation, Step S240 can prepare cellsof the target cell population for a molecular diagnostic assay, such asPCR.

In variations, the process reagent(s) can be delivered to anddistributed across the array of wells in a manner similar to that ofdistributing the biological sample or the distribution fluid at thearray in variations described for Block S210, Block S220, and/or BlockS230. In one example, as described in Section 1, a process reagent thatis stored in a cartridge of a fluid delivery module can be dispensedfrom the cartridge and dispensed into the array of wells through a fluidreservoir 160 superior and directly fluidly coupled to the array ofwells. To enhance the speed of performing various assays and processesat the array of wells, multiple process reagents can be stored (e.g.,preloaded) in the reagent cartridge of the fluid delivery module untilneeded. The fluid delivery module functions to deliver the appropriatevolume of desired reagent into the array of wells via the fluid inletand into the fluid reservoir 160, where the fluid flow velocity iscontrolled by the pumping system, to prevent damage to the cells, andimprove efficiency of the assay (e.g., the distribution of reagentswithin the wells, the distribution of the particles across the wells,etc.). Furthermore, fluid flow across the fluid reservoir 160 can befurther controlled using the upper lid of the system to create a fluidlayer at the fluid reservoir 160 to enhance the transit of the fluidacross the fluid reservoir 160 and into the wells. To dispense thedesired reagent into the fluid reservoir, Step S240 can compriserotating the cylindrical cartridge of the fluid delivery module, suchthat a chamber containing a desired reagent can be punctured by theactuation system. The process reagent can then flow into the fluidreservoir 160, to be delivered across the array of wells upon pressuregeneration by the pump of the flow control module. However, dispensingthe process reagents into the array of wells can be performed by anyother subcomponent of system 100, manually by the user, or otherwiseperformed.

In addition, Block S240 can be performed in conjunction with the thermalcontrol module to control the convective flow of the reagents across thefluid reservoir 160 and to maintain the temperature of the array ofwells (e.g., to preserve cell viability, to preserve intracellulargenetic content (e.g., mRNA, cDNA) viability, aid in diffusion ofreagents into the wells, enhance speed of flow of reagents across thefluid reservoir 160, etc.). In variations, Block S240 can includetransmitting or removing heat, through the substrate, to the cellpopulation captured at the array, which functions to provide controlledincubation and/or thermocycling of the cell population with the processreagent(s) received in variations of Block S240. Heating preferablyincludes providing uniform heating and/or cooling at each well of theset of wells of the array; however, heating can alternatively includeproviding heating and/or cooling non-uniformly across the array (e.g.,providing heat with a gradient to examine effects of different heatingparameters on the cell population). In variations, heating can includecontacting the substrate with at least one heating element, adjusting anenvironmental temperature of the substrate, and/or transmitting heatthroughout the substrate by way of heating elements coupled to orembedded within the substrate. In one example, one or more heatingelements can be coupled to the base of the substrate below the array ofwells (FIGS. 31A-31C), which functions to provide rapid heating and/orcooling to the array of wells. The system can additionally and/oralternatively include one or more heating elements embedded within anupper lid of the substrate, which functions to modulate the temperatureof the process reagents flowing through the fluid path of the fluidreservoir and enhance access to the interior of the wells (FIGS. 15A,15B, and FIG. 16). However, transmitting and/or removing heat to thearray of wells can additionally or alternatively be performed in anyother suitable manner. In a variation wherein transmitting heat includesincubating the substrate, with the cell population and a process reagentfor a desired amount of time at a desired temperature, transmitting heatcan facilitate one or more of: lysing the cell population, fixing thecell population, permeabilizing the cell population, staining the cellpopulation, performing immunochemistry for the cell population, bindinga probe to intracellular nucleic acid content of the cell population,performing an in-situ hybridization assay (e.g., a fluorescence in-situhybridization assay, FISH), performing polymerase chain reaction fornucleic acid content of the cell population, culturing the cellpopulation, and any other suitable application. However, receiving theprocess reagent(s) and/or modulating the flow (volume, velocity,temperature, etc.) of the process reagent(s) can additionally oralternatively be performed in any other suitable manner for any othersuitable application.

In one variation of Block S240, the process reagent can be a lysingreagent that releases at least one biomolecule, such as nucleic acidcontent (e.g., mRNA) and/or protein, from the population of targetcells. Lysing the captured cells is achieved by adding the lysingreagent to the array of wells at a temperature below 15° C., followed byincubating the array of wells at approximately 25° C., and/or atslightly elevated temperature such as 50° C., to complete the reaction.In some variations, lysing the cells can include distributing a layer ofoil (or other solution immiscible in the solution contained within thewell cavities) over the top of the wells prior to performing the lysingreaction, to prevent released content from transferring to adjacentwells in the array of wells. In another variation, lysing the cells caninclude distributing a layer of air over the top of the wells prior toperforming the lysing reaction, to prevent released content fromtransferring to adjacent wells in the array of wells.

In a preferred application utilizing the cell-particle pairs capturedwithin wells of the array in the ideal state, the temperature of thearray of wells can be again reduced to below 15° C. (approximately 5° C.to preserve the mRNA) after the lysing reaction, and biomoleculesreleased from each of the lysed target cells can hybridize to the probesof the corresponding particles (e.g., particles colocalized within thesame well as the target cell, associated with the cell-particle pair),forming a set of genetic complexes 70 (mRNA from the cell hybridizedwith nucleotide sequences of the set of probes) associated with eachcaptured cell. Preferably, as shown in FIG. 23, each genetic complex ofthe set of genetic complexes 70 can include at least a probe and abiomolecule (e.g., mRNA) derived from the target cell, wherein a singleset of genetic complexes 70 (e.g., all associated with biomoleculesderived from the same cell) is contained within each individual well ofthe array of wells. Generating the set of genetic complexes 70 can beachieved when each particle of a population of particles includes a setof probes 36, each probe including a unit identifier (e.g., a uniquenucleotide sequence for each particle) as well as a biomoleculeidentifier (e.g., a unique nucleotide sequence for each probe, UMI), asfurther described in Block S220. Upon cell lysis, multiple moleculesreleased from a single target cell can be identified as originating fromthe cell of the cell-particle pair by binding and/or hybridizing eachindividual biomolecule to a respective probe of the set of probes 36 ofthe particle (e.g., complementary nucleotide pairing), thereby labelingeach biomolecule with a unique nucleotide sequence including the unitidentifier and the biomolecule identifer that can be easily identifiedduring downstream processing (e.g., RNA, DNA sequencing). Thus, geneticcomplexes with the same unit identifier in the nucleotide sequence canbe considered to have been derived from the same target cell in thecell-particle pair, enabling individual analysis of multiple moleculesfrom the same cell to occur in parallel. In order to reduce non-specificlabeling of mRNAs to individual probes, various steps includingoptimizing buffer wash speed, setting an optimal binding time to captureall mRNA transcripts within an individual well, on-chip cooling duringlysis and hybridizing steps, and increasing probe density can be used.Additionally and/or alternatively, specific molecules and/or particlescan be added to the post-lysis wash solution to capture unbound cellularcontent egressed from a cell, thereby preventing unbound cellularcontent from diffusing into a neighboring well. However, the set ofgenetic complexes 70 can be otherwise configured and can include anysuitable subcomponent in association with any biomolecule, cell,particle, and/or well of the array of wells.

In an alternative embodiment of method 200, as shown in FIG. 24, FIG.25, and FIG. 26, a set of probes 36 can additionally and/oralternatively be immobilized to the interior surface 130 of individualwell cavities, left free-floating in solution within individual wells,and/or configured in any suitable manner. In one variation, biomoleculesreleased from a target cell can bind to the set of probes 36 withoutco-localization of a single particle and a single cell within the well.Specifically, particles can be distributed to the wells insingle-particle format in a method similar to that described for BlockS220 and Block S230 prior to capturing cells as described in Block S210,wherein each individual particle includes a set of probes 36 containinga common unit identifier. Rather than receiving the population ofparticles to generate a cell-particle pair in each well, receiving thepopulation of particles can result in an output condition wherein singleparticles are captured within each well. In a preferred application ofthis embodiment of method 200′, single-particle capture within each wellprior to single-cell capture can enable delivery of the sets of probesto each individual well (FIG. 26). Upon single-particle capture, the setof probes 36 can be detached from their associated particle and releasedinto individual well cavities, thereby permitting the contents of eachwell to be identifiable by the unit identifier of the set of particlestherein. Each set of probes 36 that has been released is preferablyimmobilized to an interior surface 130 of the well cavity 128 (e.g., viabiochemical bond, antibody-antigen reactivity), effectively labelingeach well with the unit identifier and allowing the particles to beremoved from the wells, thus permitting the array of wells to beaccessible by a population of target cells. Following the addition ofthe sets of probes to individual well cavities, a population of targetcells can be distributed to the wells, as described in Block S210,whereby target cells can be captured in single-cell format (e.g., onecell to each well) (FIG. 25). Upon single cell capture, cell lysis canbe performed Page io8 of 126 as described above, wherein the set ofgenetic complexes 70 comprising the released biomolecules and the set ofprobes 36 are contained within each well and coupled to the interiorsurface 130 of the well cavity 128, rather than the particle). In thisway, directly labeling each well with the unit identifier can increasethe speed and efficiency of generating sets of genetic complexes from apopulation of target cells. However, the contents of the array of wells,including genetic complexes, cells, biomolecules, and/or non-cellparticles, can be processed in any other suitable manner.

In a second variation of Block S240, process reagents can be added tothe array of wells to perform cDNA synthesis of genetic material (e.g.,the genetic complexes) of the population of target cells, therebygenerating a genetic product 80 associated with each individual cellthat can be further analyzed downstream (e.g., genetic sequencing).Synthesis of cDNA from genetic complexes containing mRNA via reversetranscription can be performed by adding a series of process reagents tothe array of wells in a predetermined sequence, and is preferablyautomated by at least one subcomponent of the system 100. In a preferredvariation, the series of process reagents can be added to the array ofwells at a set time, volume, velocity, and temperature using a programexecuted by a processor of the fluid delivery module. The method canfurther comprise receiving information regarding a sample preparationprotocol in Block S242. Block S242 is preferably performed before BlockS240, such that an automated system can be prepared to process andanalyze a biological sample based upon the information. For example,Block S242 can enable automatic alignment of reagent chambers of a setof reagent chambers of a fluid delivery module, with a fluid reservoir160 configured to deliver processing reagents into a array of wells. Ina specific example, a sequence of alignment commands can be generatedthat control rotation of a cylindrical cartridge containing isolatedprocessing reagents, thereby automating processing of the biologicalsample according to the sample preparation protocol. Block S242 canalternatively be performed before or after any suitable step of themethod 200, and can be used in coordination with information collectedfrom any other step of method 200 and/or system 100. For instance, BlockS242 can allow a user to input information about the sample preparationprotocol, and/or can automatically receive information about the samplepreparation protocol using the imaging subsystem 194, as describedpreviously in Block S216. Block S242 can, however, include any othersuitable method of receiving information regarding the samplepreparation protocol. However, the series of process reagents can beadded to the array of wells manually or by any other suitable method toperform any other suitable biochemical process.

2.5 Method—Removing Genetic Material from the Array of Wells

Block S250 recites: removing genetic material from the array of wells,which functions to collect the genetic material from the wells fordownstream processing. In variations, genetic material includingportions of genetic complexes and genetic products generated in BlockS240 can include any biological material containing identifyinginformation for the target cell (e.g., synthetic proteins, naturalproteins, nucleic acids, a biomarker, a byproduct of a biochemicalreaction, etc.). Prior to removal from the array of wells, the geneticmaterial can be coupled to the population of particles, floating in thesolution within the well cavity 128, attached to a region of theinterior surface 130 of the well cavity 128, or in any suitable locationwithin the well or in relation to any other suitable subcomponent withinthe well. In a first variation, the genetic material includes the rawintracellular genetic content from lysed cells (e.g., protein, mRNA,DNA, proteins). In a second variation, the genetic material includes theset of probes 36, either detached from a particle, or coupled to aparticle. In a third variation, the genetic material includes thegenetic product 80 (e.g., cDNA sequences formed by reverse transcriptionof the genetic complexes in variations of Block S240). In a fourthvariation, the genetic material includes genetic product 80 from BlockS240 that has been amplified by polymerase chain reaction (e.g., a cDNAlibrary). However, the genetic material can include any suitablebiological materials that can be removed from the array of wells andsubsequently analyzed or processed. Block S250 is performed preferablyafter Block S240 has been completed, but can be performed at any othersuitable time in relation to other steps of method 200. In anotherembodiment, the system 100 can achieve multiple cycles of Blocks S240and S250 for repeated generation and elution of similar genetic product,thereby permitting preparation of multiple libraries and/or performingsequencing from the same set of single cell-derived genetic complexes.

In a first variation wherein the genetic material remains coupled to thepopulation of particles, removal of the genetic material can be achievedby removing the population of particles from the array of wells. In onevariation, the particles can be flushed out from the array of wells byincreasing the fluid volume of an extraction fluid within the array ofwells (e.g., manually pipetting fluid, flowing a fluid into the array ofwells, etc.). In an example, an extraction fluid (e.g., 5-10 mL PBS) canbe dispensed into the array of wells in a direction perpendicular to theupper broad surface of the substrate, but can additionally and/oralternatively be dispensed into the array of wells by flowing theextraction fluid along the fluid path through the fluid reservoir in adirection parallel to the upper broad surface of the substrate, whereinthe extraction fluid can enter the array of wells through the opensurfaces of the wells by diffusion. Additionally and/or alternatively,dispensing the extraction fluid into the array of wells can includeapplying force to the extraction fluid, thereby altering the velocity ofthe extraction fluid, to reposition the particles within the wells(e.g., by pipetting up and down into the fluid reservoir, applying a netpositive or net negative pressure to the fluid, etc.). In an example,adding a sufficient volume of extraction fluid at a specific fluidvelocity (or cycles of at least a first and a second fluid velocity) canexpel particles previously submerged within the well cavity 128 throughthe open surface of the well and into the fluid reservoir, where theparticles can be collected for downstream processing (e.g., flowedthrough the outlet into a collection receptacle, directly retrieved fromthe reservoir, etc.). In a second variation, the particles can beremoved from the array of wells by inverting the substrate (e.g.,rotating the substrate about its transverse axis passing through themidpoint of the substrate by 180°) and incubating the substrate for atime period of up to 60 minutes (e.g., approximately 10, 20, 30, 40, 50,60 minutes). In variations wherein the fluid reservoir is sealed by thefirst plate 150 (as described in a previous section), particles can exitthe array of wells through the open surfaces of the wells and into thefluid reservoir by gravitational force, however, the particles canegress from the array of wells into any other suitable collectionreceptacle. In a third variation, the particles can be directlyextracted from the array of wells, using the extraction module.Additionally and/or alternatively, the speed of extraction of theparticles can be accelerated by additional steps, including:centrifuging the substrate, physically agitating the substrate (e.g.,shaking, vibrating), increasing and/or decreasing fluid volume into thearray of wells, applying a force to the particles (e.g., magnetic,electric, physical), or any other suitable method of removal.

In a second variation wherein the genetic material remains coupled tothe population of particles, genetic material can be removed byseparating the genetic product 80 from the particle within the well, andpipetting the fluid from the wells, leaving the particle within thewell. In an example, the genetic complexes are bound to the particle bya reversible biochemical bond, such as a photo-cleavable linker, whereinthe linker is reversibly attachable to the particle and can be removedby exposing the particles to specific wavelengths of light (e.g.,visible light spectrum, ultraviolet spectrum). However, variouschemistries can be used to controllably remove (or attach) the probe tothe particle, the well, or any other suitable surface for downstreamanalysis. In a specific example, uniform light originating from theoptical subsystem described in Section 1 can provide uniformillumination of the array of wells from above the substrate proximal thefirst broad face of the substrate, and into the well cavities throughthe open surfaces of the wells. In this example, ultraviolet lightranging from 300-400 nm (e.g., 365 nm) can be irradiated into the arrayof wells for up to 30 minutes (e.g., approximately 5, 10, 20, 30minutes). In a preferred variation, the thermal control module caninclude a reflective surface arranged below the substrate at the secondbroad face (lower surface), directly opposing the first broad facethrough which the ultraviolet light passes, thereby reflecting incidentlight back into the well and illuminating the entire bead for uniformand simultaneous photo-cleaving of the genetic omplexes from thepopulation of particles. However, uniform irradiation of the populationof particles contained within the wells can be achieved by any othersuitable method, including: rotating the beads within the wells bygently shaking or rotating the substrate during exposure, and usingfluid flow across the fluid reservoir 160 in conjunction with thepressure system to manipulate rotation of the beads within the wells.Furthermore, any wavelength or combination of wavelengths of light orcan be used to illuminate the population of particles to performchemistry sensitive to light. In addition, the intensity of the incidentlight beam can be of any suitable intensity. In this variation whereinoptical illumination is used to separate the genetic product 80 from thepopulation of particles within the well, the thermal control module canbe used to control the temperature of the array of wells to minimizeeffects of heating of the solution with the well cavities to preservethe quality of the genetic product. Furthermore, additional reagents canbe added to the array of wells to enhance the efficiency or aid in theperformance of photo-cleaving chemistries, such as reagents to increasespeed and/or efficiency of removal, or agents to improve viability ofgenetic material during removal.

In an alternative variation of Block S250, separating the geneticmaterial from the particle can further include, instead of removing thegenetic material from the well, removing the bare particle from thewell, thereby leaving the genetic material in individual wells forfurther processing. In this variation, the genetic material can be leftfreely floating within the well cavity 128, or can be immobilized withinthe well cavity 128. The particles can be removed by methods inpreviously described variations (e.g., magnet), leaving the geneticmaterial to couple to the interior surface 130 of the well cavity 128.As described in Section 1, the interior surface 130 of the well cavity128 can include surface features, wherein small molecules can bephysically, energetically, or chemically entrapped, and/or surfacefeatures wherein small molecules bind with high affinity (higher bindingaffinity than to the particle). In a specific example, the geneticmaterial can get physically entrapped by a set of ridges or protrusionswithin the walls of each well. In another specific example, the geneticmaterial can include a functional linker incorporated from theconjugated probe (e.g., biotin protein), and can be configured to bindto streptavidin that has been previously coated on the interior surface130 of the wells. In another specific example, the genetic material canbind to affinity microspheres/agents lining the surface of the wells.However, genetic material can be processed in any suitable manner.

Downstream analysis of captured target cells and/or genetic materialderived from captured target cells can include any one or more of:harvesting contents of the set of wells (e.g., cells, intracellularcontent), culturing cells captured at the set of wells, detectingbiomarkers exhibited by the cell population (e.g., using fluorescentdetection), performing a quantitative analysis (e.g., a quantitativeanalysis of mRNA expression), characterizing a cell phenotype (e.g., acancer cell phenotype) based upon biomarker expression, providing arecommended therapy based upon characterization of a cell phenotype,performing flow cytometry with captured cells of the cell population,and performing any other suitable analysis. In a preferred application,wherein genetic material, specifically cDNA produced in Block S240, isused for RNA sequencing, genetic material can be further processed togenerate cDNA libraries containing genetic material of specific fragmentsizes and quality by performing: exonuclease treatment,pre-amplification PCR, SPRI cleanup, and tagmentation. Furthermore, datacollected using system 100 and through implementation of variations ofmethod 200 can be used to generate and utilize single-cell targetedpanels including: genotyping libraries, CRISPR pools, immune profiling,pathway analysis, drug screening, and lineage tracing (FIG. 34).However, variations of method 200 can additionally or alternativelyinclude any other suitable steps or blocks that facilitate reception,processing, and/or analysis of the cell population in at least one ofsingle-cell format and single-cluster format.

The system 100 and method 200 of the preferred embodiment and variationsthereof can be embodied and/or implemented at least in part as a machineconfigured to receive a computer-readable medium storingcomputer-readable instructions. The instructions are preferably executedby computer-executable components preferably integrated with the systemand one or more portions of a processor and/or a controller. Thecomputer-readable medium can be stored on any suitable computer-readablemedia such as RAMs, ROMs, flash memory, EEPROMs, optical devices (CD orDVD), hard drives, floppy drives, or any suitable device. Thecomputer-executable component is preferably a general or applicationspecific processor, but any suitable dedicated hardware orhardware/firmware combination device can alternatively or additionallyexecute the instructions.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, or portion of code, whichcomprises one or more executable instructions for implementing thespecified logical function(s). It should also be noted that, in somealternative implementations, the functions noted in the block can occurout of the order noted in the FIGURES. For example, two blocks shown insuccession may, in fact, be executed substantially concurrently, or theblocks may sometimes be executed in the reverse order, depending uponthe functionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts, or combinations of special purpose hardware andcomputer instructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

We claim:
 1. A method for isolating and analyzing a set of target cellscomprising: receiving the set of target cells into an array of wellsdefined at a surface plane of a substrate, wherein each well in thearray of wells extends perpendicular to and below the surface plane intothe substrate; achieving a particle-accessible state comprising the setof target cells captured in single-cell format at a first subset ofwells of the array of wells comprising at least a first well, wherein afirst target cell of a set of target cells is received into an open endof the first well; distributing a set of particles toward the array ofwells, wherein each particle of the set of particles is coupled to aprobe having a binding affinity for a target component associated withone or more of the set of target cells; upon distributing the set ofparticles toward the array of wells, achieving a desired state for thefirst well in the particle-accessible state, wherein achieving thedesired state comprises co-capturing a first particle of the set ofparticles with the first target cell within the first well; processing aset of desired wells of the array of wells comprising at least the firstwell, wherein processing the set of desired wells comprises delivering aprocess reagent to the set of desired wells; and providing communicationbetween target material of the set of desired wells and a thermalcontrol module, for at least one of controlled heating and controlledcooling of target material processed using the set of desired wells. 2.The method of claim 1, wherein providing communication between targetmaterial of the set of desired wells and a heat transmitting subsystemcomprises delivering target material of the set of desired wells fromthe set of desired wells, by way of the set of particles, into a processcontainer; and establishing thermal communication between the processcontainer and the thermal control module.
 3. The method of claim 1,wherein providing communication between target material of the set ofdesired wells and a heat transmitting subsystem comprises establishingthermal communication between the substrate and the thermal controlmodule.
 4. The method of claim 1, wherein the probe comprises particlelinker region configured to couple to at least one of the set ofparticles, a primer sequence, a cell barcode sequence, and a uniquemolecular identifier region.
 5. The method of claim 1, wherein thetarget material comprises cDNA, and wherein the method further comprisesgenerating a cDNA library associated with the set of target cells uponperforming an exonuclease treatment process and a polymerase chainreaction process with the target material, in cooperation with thethermal control module.
 6. The method of claim 1, further comprising:achieving a particle-saturated state for a second well of the array ofwells, wherein achieving the particle-saturated state comprisesreceiving at least a second particle and a third particle of the set ofparticles into the second well, wherein the third particle traverses thesurface plane; and redistributing the third particle to a third well ofthe array of wells, thereby transitioning the second well from theparticle-saturated state to a desired state comprising the second targetcell co-localized with the second particle.
 7. The method of claim 6,wherein re-distributing the third particle comprises: receiving a fluidinto a fluid reservoir spanning the array of wells along the surfaceplane and in a direction parallel to the surface plane, therebytransferring the third particle from the second well and into the thirdwell.
 8. The method of claim 1, wherein delivering the process reagentto the set of desired wells comprises delivering at least one of a celllysis reagent and an activation enzyme configured for probehybridization to target material derived from the set of target cells.9. The method of claim 1, wherein distributing the set of particlestoward the array of wells comprises: with a flow control module coupledto a fluid reservoir spanning the array of wells, controlling a flowdirection of the set of particles within a particle distribution fluid,wherein the flow direction alternates between a first direction and asecond direction opposing the first direction.
 10. A method forisolating and analyzing a set of target cells comprising: receiving theset of target cells into an array of wells defined at a surface plane ofa substrate, wherein each well in the array of wells extendsperpendicular to and below the surface plane into the substrate;achieving a particle-accessible state for a first subset of wells of thearray of wells comprising at least a first well and a second well,wherein a first target cell of a set of target cells is received into anopen end of the first well, and a second target cell of the set oftarget cells is received into an open end of the second well;distributing a set of particles toward the array of wells, wherein eachparticle of the set of particles is coupled to a probe having a bindingaffinity for a target component associated with one or more of the setof target cells; upon distributing the set of particles toward the arrayof wells, achieving a desired state for the first well in theparticle-accessible state, wherein achieving the desired state comprisesco-capturing a first particle of the set of particles with the firsttarget cell within the first well; and achieving a particle-saturatedstate for the second well in the particle accessible state, whereinachieving the particle-saturated state comprises receiving at least asecond particle and a third particle of the set of particles into thesecond well, wherein the third particle traverses the surface plane;re-distributing the third particle to a third well of the array ofwells, wherein re-distributing the third particle comprises: receiving afluid into a fluid reservoir spanning the array of wells along thesurface plane and in a direction parallel to the surface plane, therebytransitioning the second well from the particle-saturated state to adesired state comprising the second target cell co-captured with thesecond particle; processing a set of desired wells of the array of wellscomprising at least the first well and the second well, whereinprocessing the set of desired wells comprises delivering a processreagent to the set of desired wells; and transmitting target materialfrom the set of desired wells by way of the set of particles.
 11. Themethod of claim 10, wherein transmitting target material comprisesdelivering the target material from the array of wells into a processcontainer; and establishing thermal communication between the processcontainer and a thermal control module for at least one of controlledheating and controlled cooling of the target material.
 12. The method ofclaim 10, wherein the target component is a ribonucleic acid and theprobe of each particle in the set of particles comprises a nucleotidesequence configured to bind to a portion of the ribonucleic acid. 13.The method of claim 12, wherein the probe of each particle in the set ofparticles comprises particle linker region configured to couple to atleast one of the set of particles, a primer sequence, a cell barcodesequence, and a unique molecular identifier region.
 14. The method of10, further comprising: within the first well of the set of desiredwells, performing a biochemical process with the target component. 15.The method of claim 13, wherein performing the biochemical processcomprises performing reverse transcription within the first well of theset of desired wells, thereby producing, within the first well, a firstnucleotide sequence associated with a first genetic complex.
 16. Themethod of claim 10, wherein delivering the process reagent comprisesdelivering the process reagent into the array of wells and maintaining atemperature of contents of the array of wells below 15° C. with athermal control module coupled to the substrate.
 17. The method of claim10, further comprising establishing thermal communication between thesubstrate and a thermal control module, and modulating temperature ofcontents of the array of wells between 5° C. and 95° C. with the thermalcontrol module.
 18. The method claim 17, further comprising performing adownstream process with the target material, wherein the target materialcomprises cDNA, and wherein the method further comprises generating acDNA library associated with the set of target cells upon performing anexonuclease treatment process and a polymerase chain reaction processwith the target material, in cooperation with the thermal controlmodule.
 19. The method of claim 10, wherein delivering the processreagent to the set of desired wells comprises delivering at least one ofa cell lysis reagent and an activation enzyme configured for probehybridization to target material derived from the set of target cells.20. The method of claim 10, wherein receiving the set of target cellsinto the array of wells comprises providing the array of wells, eachwell in the array of wells having a length between 20 and 75 micrometersand a width between 20 and 30 micrometers.